U.S. patent number 10,544,456 [Application Number 15/655,616] was granted by the patent office on 2020-01-28 for systems and methods for nucleic acid sequencing.
This patent grant is currently assigned to GENAPSYS, INC.. The grantee listed for this patent is GenapSys, Inc.. Invention is credited to Meysam R. Barmi, Hesaam Esfandyarpour, Paul Kenney, Ali Nabi, Saurabh Paliwal, Kosar Parizi, Hamid Rategh, Seth Stern.
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United States Patent |
10,544,456 |
Esfandyarpour , et
al. |
January 28, 2020 |
Systems and methods for nucleic acid sequencing
Abstract
Provided herein are systems and methods for processing and
analyzing nucleic acids and other biomolecules. Methods may include
processing nucleic acid molecules in an emulsion of droplets.
Methods of analyzing nucleic acid molecules may include coupling
nucleic acids to a bead or other support. Methods may include
analysis of nucleic acid molecules using a redox mediator. In some
cases, analysis of the nucleic acid molecule includes determining a
nucleotide sequence of the nucleic acid molecule.
Inventors: |
Esfandyarpour; Hesaam (Redwood
City, CA), Parizi; Kosar (Redwood City, CA), Paliwal;
Saurabh (Mountain View, CA), Stern; Seth (Menlo Park,
CA), Kenney; Paul (Sunnyvale, CA), Barmi; Meysam R.
(Menlo Park, CA), Nabi; Ali (Belmont, CA), Rategh;
Hamid (Cupertino, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
GenapSys, Inc. |
Redwood City |
CA |
US |
|
|
Assignee: |
GENAPSYS, INC. (Redwood City,
CA)
|
Family
ID: |
60996038 |
Appl.
No.: |
15/655,616 |
Filed: |
July 20, 2017 |
Prior Publication Data
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|
|
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Document
Identifier |
Publication Date |
|
US 20180100190 A1 |
Apr 12, 2018 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62364489 |
Jul 20, 2016 |
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62375197 |
Aug 15, 2016 |
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62418101 |
Nov 4, 2016 |
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62444700 |
Jan 10, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6869 (20130101); C12Q 1/6869 (20130101); C12Q
2563/116 (20130101); C12Q 2563/149 (20130101); C12Q
2565/607 (20130101) |
Current International
Class: |
C12Q
1/6869 (20180101) |
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|
Primary Examiner: Bhat; Narayan K
Attorney, Agent or Firm: Wilson Sonsini Goodrich &
Rosati
Parent Case Text
CROSS-REFERENCE
This application claims the benefit of U.S. Provisional Patent
Application No. 62/364,489, filed Jul. 20, 2016, U.S. Provisional
Patent Application No. 62/375,197, filed Aug. 15, 2016, U.S.
Provisional Patent Application No. 62/418,101, filed Nov. 4, 2016,
and U.S. Provisional Patent Application No. 62/444,700, filed Jan.
10, 2017, each of which is entirely incorporated herein by
reference.
Claims
What is claimed is:
1. A method for sequencing a nucleic acid template, comprising: (a)
contacting a nucleic acid template with a sensing fluid containing
a population of nucleotides, wherein said nucleic acid template is
hybridized to a primer that is coupled to a bead, which bead is
positioned proximate to a sensor in a sensor array, wherein said
sensor comprises at least two electrodes, wherein said sensing
fluid has a sensing fluid bulk conductivity and a surface of said
bead has a surface conductivity to provide a Dukhin number that is
less than 1 such that (i) a conductivity measurement by said at
least two electrodes with said bead positioned proximate to said
sensor is substantially similar to (ii) another conductivity
measurement by said at least two electrodes without said bead
positioned proximate to said sensor; (b) using said at least two
electrodes of said sensor to detect a conductivity change within a
Debye layer of said bead upon incorporation of at least one
nucleotide of said population of nucleotides into a growing nucleic
acid strand, which growing nucleic acid strand is derived from said
primer and is complementary to said nucleic acid template, which
conductivity change is detected based at least in part on an
electrical current change through said Debye layer; (c) washing
said sensor array to remove unincorporated nucleotides of said
population of nucleotides from said sensor array; and (d) repeating
(a)-(c) to obtain sequence information for said nucleic acid
template.
2. The method of claim 1, wherein an electrode of said at least two
electrodes is exposed to said sensing fluid.
3. The method of claim 1, wherein (b) further comprises detecting a
change in impedance within said Debye layer of said bead upon
incorporation of said at least one nucleotide.
4. The method of claim 3, wherein said change in impedance within
said Debye layer is detected at steady state.
5. The method of claim 1, wherein said at least two electrodes are
positioned within said Debye layer of said bead.
6. The method of claim 1, wherein said sensing fluid has a solute
concentration between about 0.15 millimolar and about 6
millimolar.
7. The method of claim 1, further comprising, prior to (b): (i)
contacting said sensor array with a probe fluid, wherein said probe
fluid has a probe fluid bulk conductivity that is at least about 50
times greater than or at least about 50 times less than said
surface conductivity of said surface of said bead; and (ii) using
said sensor to detect signals that are indicative of a presence of
said bead in proximity to said sensor.
8. The method of claim 7, wherein an additional Dukhin number
determined from said probe fluid bulk conductivity and said surface
conductivity of said surface of said bead is substantially less
than 1.
9. The method of claim 7, wherein an additional Dukhin number
determined from said probe fluid bulk conductivity and said surface
conductivity of said surface of said bead is substantially greater
than 1.
10. The method of claim 7, wherein (b), (c), and (d) are performed
only at sensors of said sensor array at which signals indicative of
bead occupancy are observed.
Description
BACKGROUND
The human genome has created interest in technologies for rapid
nucleic acid analysis, including nucleic acid sequencing, both for
small and large-scale applications. Presently available nucleic
acid sequencing technologies include detection of fluorescent
nucleotides; detection of proton byproducts of polymerase activity;
and (iii) detection of currents through nanopores. In the context
of sequencing, important considerations include accuracy, speed,
read length, cost, instrument size and complexity, and the amount
of nucleic acid template required to generate sequencing
information. Unfortunately, large-scale genome projects often
remain too costly and/or infeasible, due to shortcomings in
available sequencing technologies. Available sequencing
technologies, such as those mentioned above, often have sample
preparation, accuracy and/or scalability issues that present
significant challenges their mainstream implementation.
SUMMARY
Recognized herein is the need for improved systems and methods for
sensing biological reactions, including nucleic acid sequencing
reactions. Systems and methods provided herein may have utility in
sequencing nucleic acids associated with beads or nucleic acids
associated with polymer films. In some cases, signals derived from
the sequencing reaction are detected at a buffer composition and
concentration that reduces the sensitivity of the system to
movements of a bead relative to one or more sensors that detect
such signals.
The present disclosure provides methods and systems for sample
analysis or identification, such as nucleic acid sequencing. The
present disclosure provides methods and systems that may enable
sample preparation and identification (e.g., sequencing) without
the use of particles, such as beads. This may enable a sample to be
prepared and identified at substantially reduced cost and
complexity as compared to other systems and methods.
The disclosure also provides systems and methods for improved
nucleic acid analysis that overcome status-quo deficiencies and
permit low-cost, scalable nucleic acid sequencing technologies. As
knowledge of the genetic basis for human diseases increases, there
will be an ever-increasing need for accurate nucleic acid analysis
tools that are accurate, affordable and scalable for clinical
applications. The present disclosure provides systems and methods
that make use of redox mediator moieties that are detectable using
electronic sensors to perform nucleic acid sequencing.
In an aspect, the present disclosure provides methods for
sequencing a nucleic acid template comprising: (a) contacting a
nucleic acid template with a sensing fluid containing a population
of nucleotides, wherein the nucleic acid template is hybridized to
a primer that is coupled to a bead, which bead is positioned
proximate to a sensor in a sensor array, wherein the sensor
comprises at least two electrodes, and wherein the sensing fluid
has a bulk conductivity and a surface of the bead has a surface
conductivity to provide a Dukhin number that is less than about 1;
(b) using the sensor to detect a change in conductivity within a
Debye layer of the bead upon incorporation of at least one
nucleotide of the population of nucleotides into a growing nucleic
acid strand, which growing nucleic acid strand is derived from the
primer and is complementary to the nucleic acid template; (c)
washing the sensor array to remove unincorporated nucleotides of
the population of nucleotides from the sensor array; and (d)
repeating (a)-(c) to obtain sequence information for the nucleic
acid template.
In some embodiments, a given electrode of the at least two
electrodes is exposed to the sensing fluid. In some embodiments,
(b) further comprises detecting a change in impedance within the
Debye layer of the bead upon incorporation of the at least one
nucleotide. In some embodiments, the change in impedance within the
Debye layer is detected at steady state. In some embodiments, the
at least two electrodes are positioned within the Debye layer of
the bead. In some embodiments, the sensing fluid has a solute
concentration between about 0.15 millimolar and about 6
millimolar.
In some embodiments, the method further comprises, prior to (b):
(i) contacting the sensor array with a probe fluid, wherein the
probe fluid has a bulk conductivity that is at least about 50 times
greater than or 50 times less than the conductivity associated with
the surface of the bead; and (ii) using the sensor to detect
signals that are indicative of a presence of the bead in proximity
to the sensor. In some embodiments, a Dukhin number determined from
the bulk conductivity of the probe fluid and the conductivity of
the surface of the bead is substantially less than 1. In some
embodiments, a Dukhin number determined from the bulk conductivity
of the probe fluid and the conductivity of the surface of the bead
is substantially greater than 1. In some embodiments, (b), (c), and
(d) are performed only at sensors of the sensor array at which
signals indicative of bead occupancy are observed.
In an aspect, the present disclosure provides methods for
determining bead occupancy at sites of a sensor array, comprising:
(a) contacting a sensor array with a plurality of beads, wherein
the sensor array comprises a plurality of sensors each having at
least two electrodes, to provide a given bead of the plurality of
beads at a given position in proximity to an individual sensor of
the plurality of sensors; (b) contacting the sensor array with a
probe fluid that has a bulk conductivity that is at least about 50
times greater than or 50 times less than a conductivity associated
with a surface of the given bead; (c) using the individual sensor
to detect signals that are indicative of a presence of the given
bead in proximity to the sensor; and (d) identifying the given
position of the sensor array as occupied by the given bead.
In some embodiments, the probe fluid has a concentration of solutes
between about 0.01 millimolar and about 1 molar. In some
embodiments, a Dukhin number determined from the bulk conductivity
of the probe fluid and the conductivity of the surface of the bead
is substantially less than 1. In some embodiments, a Dukhin number
determined from the bulk conductivity of the probe fluid and the
conductivity of the surface of the bead is substantially greater
than 1. In some embodiments, the method further comprises a nucleic
acid coupled to the given bead. In some embodiments, the signals
comprise electrical current. In some embodiments, a given electrode
of the individual sensor is positioned within a Debye layer of the
given bead.
In an aspect, the present disclosure provides methods for
processing a nucleic acid sample, comprising: (a) providing a
mixture comprising a first set of droplets and a second set of
droplets, wherein a first droplet of the first set of droplets
comprises (i) a bead, (ii) a recombinase, (iii) a polymerizing
enzyme, and (iv) a nucleic acid molecule from the nucleic acid
sample, and wherein a second droplet of the second set of droplets
comprises an activating agent that increases a rate at which the
recombinase processes the nucleic acid molecule to permit the
primer to hybridize to the nucleic acid molecule to conduct a
primer extension reaction in presence of the polymerizing enzyme,
to generate an amplification product(s) from the nucleic acid
molecule; (b) merging the first droplet with the second droplet in
the mixture to generate a third droplet as part of a third set of
droplets, wherein the third droplet comprises the bead having
coupled thereto the nucleic acid molecule, recombinase, primer and
polymerizing enzyme; and (c) conducting the primer extension
reaction to generate the amplification product(s) from the nucleic
acid molecule in the third droplet.
In some embodiments, the first droplet further comprises buffer,
salts, crowding agents, dNTPs, primers, or any combination thereof.
In some embodiments, the second droplet further comprises primers,
dNTPs, ATP, recombinase loading enzyme, single-stranded DNA-binding
protein, an ATP-regenerating unit, buffer, salt, and crowding
agents. In some embodiments, the activating agent is a magnesium
salt. In some embodiments, formation of the third droplet comprises
subjecting the mixture to low speed stirring or shaking. In some
embodiments, the primer extension reaction occurs under isothermal
conditions.
In some embodiments, the method further comprises directing the
third set of droplets through a set of obstacles to control a shape
or a size of each droplet of the third set of droplets. In some
embodiments, the set of obstacles have a comb-like structure. In
some embodiments, the shape or the size of each droplet of the
third set of droplets is controlled by flowrate, pressure, obstacle
shape, and obstacle size.
In some embodiments, the method further comprises disrupting the
third set of droplets with a disrupting mixture comprising an
emulsion disruptor and a deactivating agent, wherein disrupting the
third set of droplets forms a homogenous solution. In some
embodiments, the method further comprises capturing multiple of the
bead coupled to amplification product(s) with a capture bead to
form a multi-bead complex. In some embodiments, the capture bead
exclusively binds to the bead coupled to amplification product(s).
In some embodiments, the multi-bead complex is appreciably larger
than a non-complexed bead. In some embodiments, the method further
comprises directing the beads through a set of obstacles to
separate the multi-bead complex from the non-complexed bead via
size selection.
In an aspect, the present disclosure provides methods for
sequencing a nucleic acid molecule, comprising: (a) activating a
sensor comprising a support comprising at least two electrodes and
a polymeric material adjacent to the support, wherein the at least
two electrodes are exposed to a solution comprising the polymeric
material, wherein the polymeric material retains the nucleic acid
molecule during a sequencing reaction; (b) subjecting the nucleic
acid molecule to the sequencing reaction to yield signals
indicative of individual bases of the nucleic acid molecule; (c)
during the sequencing reaction, using the at least two electrodes
of the sensor to detect the signals; and (d) using the signals
detected in (c) to generate a sequence of the nucleic acid
molecule.
In some embodiments, the at least two electrodes comprise a
chemically inert conducting material. In some embodiments, the
support comprises a surface modified silicon oxide or a metal
oxide. In some embodiments, the polymeric material is coupled to
the surface modified silicon oxide or metal oxide. In some
embodiments, the polymeric material bridges or covers the at least
two electrodes.
In some embodiments, the polymeric material is a hydrogel. In some
embodiments, the hydrogel comprises reactive and non-reactive
co-monomers. In some embodiments, the polymeric material is a
porous polymer monolith. In some embodiments, the porous polymer
monolith is a homopolymer, copolymer, or terepolymer comprising
reactive functional groups.
In some embodiments, the polymeric material is seeded with primers
and wherein the primers are reactively coupled to the polymeric
material. In some embodiments, the primers participate in an
amplification reaction to form clonal colonies. In some
embodiments, the primers are seeded at a concentration ranging from
1 picomolar to 4000 picomolar. In some embodiments, the
amplification reaction is invader amplification, bridge
amplification, wildfire amplification, recombinase polymerase
amplification, or polymerase chain reaction with a confinement
approach.
In some embodiments, during the sequencing reaction, the at least
two electrodes are coupled to a Debye layer having of the nucleic
acid molecule. In some embodiments, the incorporation of
nucleotides into the nucleic acid molecule is performed within the
Debye layer. In some embodiments, the signals indicative of the
individual bases are electrochemical signals. In some embodiments,
the signals indicative of the individual bases are impedance
signals. In some embodiments, the signals indicative of the
individual bases are detected during steady state conditions.
In an aspect, the present disclosure provides a system for
sequencing a nucleic acid molecule, comprising: a sensor comprising
a support comprising at least two electrodes and a polymeric
material adjacent to the support, wherein during use the at least
two electrodes are exposed to a solution comprising the polymeric
material, wherein the polymeric material retains the nucleic acid
molecule during a sequencing reaction, which sequencing reaction
yields signals indicative of individual bases of the nucleic acid
molecule; and one or more computer processors operatively coupled
to the sensor, wherein the one or more computer processors are
programmed to (i) subject the nucleic acid molecule to the
sequencing reaction to yield the signals indicative of the
individual bases of the nucleic acid molecule; (ii) during the
sequencing reaction, use the at least two electrodes of the sensor
to detect the signals; and (iii) use the signals detected in (ii)
to generate a sequence of the nucleic acid molecule.
In some embodiments, the at least two electrodes comprise a
chemically inert conducting material. In some embodiments, the
support comprises a surface modified silicon oxide or a metal
oxide. In some embodiments, the polymeric material is coupled to
the surface modified silicon oxide or metal oxide. In some
embodiments, the polymeric material bridges or covers the at least
two electrodes.
In some embodiments, the polymeric material is a hydrogel. In some
embodiments, the hydrogel comprises reactive and non-reactive
co-monomers. In some embodiments, the polymeric material is a
porous polymer monolith. In some embodiments, the porous polymer
monolith is a homopolymer, copolymer, or terepolymer comprising
reactive functional groups.
In some embodiments, the polymeric material is seeded with primers
and the primers are reactively coupled to the polymeric material.
In some embodiments, the primers participate in an amplification
reaction to form clonal colonies. In some embodiments, the primers
are seeded at a concentration ranging from about 1 picomolar to
about 4000 picomolar. In some embodiments, the amplification
reaction is invader amplification, bridge amplification, wildfire
amplification, recombinase polymerase amplification, or polymerase
chain reaction with a confinement approach.
In some embodiments, during the sequencing reaction, the at least
two electrodes are coupled to a Debye layer of the nucleic acid
molecule. In some embodiments, the incorporation of nucleotides
into the nucleic acid molecule is performed within the Debye layer.
In some embodiments, the signals indicative of the individual bases
are electrochemical signals. In some embodiments, the signals
indicative of the individual bases are impedance signals. In some
embodiments, the signals indicative of the individual bases are
detected during steady state conditions.
In an aspect, the present disclosure provides methods for
sequencing a nucleic acid molecule, the method comprising: (a)
tethering a template nucleic acid molecule to a sensor or a surface
in proximity to the sensor; (b) creating an elongation complex
tethered to the sensor or the surface in proximity to the sensor,
wherein the elongation complex comprises (i) a nucleic acid
polymerase associated with the template nucleic acid molecule and
(ii) an oligonucleotide hybridized to the template nucleic acid
molecule; (c) contacting the elongation complex with a solution
comprising nucleotides under conditions sufficient to associate a
nucleotide complimentary to the template nucleic acid molecule with
the elongation complex, wherein a given nucleotide of the
nucleotides is coupled to a redox mediator moiety; (d) using the
sensor to detect a signal indicative of the redox mediator moiety
when the nucleotide is associated with the elongation complex; (e)
incorporating the nucleotide into the oligonucleotide to subject
the redox mediator moiety to release from the nucleotide; and (f)
repeating (c)-(e), thereby sequencing the template nucleic acid
molecule.
In some embodiments, a plurality of clonal template nucleic acid
molecules is tethered to the sensor or the surface in proximity to
the sensor. In some embodiments, the plurality of clonal template
nucleic acid molecules is generated with the aid of polymerase
walking. In some embodiments, the elongation complex is tethered to
the sensor or the surface in proximity to the sensor via the
polymerase. In some embodiments, in each iteration of (c), the
solution comprises only one of the nucleotides adenine (A),
cytosine (C), guanine (G), uracil (U) or thymine (T), or a variant
thereof, and each iteration of (c) contacts the elongation complex
with a different nucleotide.
In some embodiments, the redox mediator moiety comprises an organic
compound, an organometallic compound, a nanoparticle, or a metal.
In some embodiments, a plurality of redox mediator moieties are
bound to the nucleotide. In some embodiments, the elongation
complex is tethered to the sensor or the surface in proximity to
the sensor via a binding pair. In some embodiments, the binding
pair is a biotin-streptavidin binding pair. In some embodiments,
the redox mediator moiety is attached to a phosphate of the
nucleotide. In some embodiments, the nucleotide is associated with
the elongation complex for a time period between about 10 and about
500 milliseconds (ms). In some embodiments, the sensor is among a
plurality of sensors and wherein a given one of the plurality of
sensors is individually addressable.
Additional aspects and advantages of the present disclosure will
become readily apparent to those skilled in this art from the
following detailed description, wherein only illustrative
embodiments of the present disclosure are shown and described. As
will be realized, the present disclosure is capable of other and
different embodiments, and its several details are capable of
modifications in various obvious respects, all without departing
from the disclosure. Accordingly, the drawings and description are
to be regarded as illustrative in nature, and not as
restrictive.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in
this specification are herein incorporated by reference to the same
extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings (also "figure" and
"FIG." herein), of which:
FIG. 1 schematically depicts an example integrated sequencing
platform;
FIGS. 2A-2F show example sensor arrays and sensor use; FIG. 2A
shows a schematic of an example sensor array; FIG. 2B shows a
schematic of an example sensor array with beads carrying nucleic
acids, which beads are immobilized to the sensor array; FIG. 2C
shows a schematic of an example sensor array with beads carrying
nucleic acids and immobilized to the sensor array in contact with
reagents suitable for nucleic acid amplification; FIG. 2D shows a
schematic of an example sensor array where nucleic acid
amplification occurs on beads positioned at various sensors of the
sensor array; FIG. 2E shows a schematic example of removing
reagents from an example sensor array; FIG. 2F shows a schematic of
an example sensor array where nucleic acids are sequenced at
various positions of the sensor array;
FIG. 3 shows a an example detection device;
FIG. 4 shows an example computer system that is programmed or
otherwise configured to control, regulate or implement devices,
systems and methods described herein;
FIG. 5 schematically depicts an example bead-sensor structural
configuration;
FIG. 6 schematically depicts an example of conductivity at a bead
surface and in a bulk fluid adjacent to the bead surface;
FIG. 7 graphically depicts an example of the effect of buffer
concentration on the sensitivity of observed current to movement of
a bead associated with a sensor;
FIG. 8 graphically depicts an example of bead occupancy on an
example sensor array;
FIG. 9 graphically depicts example data relating to bead occupancy
on example sensor arrays;
FIG. 10 schematically illustrates a method for processing a nucleic
acid sample;
FIGS. 11A-11F schematically illustrate obstacle based
emulsification and arrays thereof.
FIG. 11A shows droplets flowing towards an obstacle; FIG. 11B shows
droplet distorting around the object due to shear forces; FIG. 11C
shows a droplet splitting around the obstacle; FIG. 11D shows
obstacles may have an arbitrary shape; FIG. 11E shows obstacles may
be confined in a flow cell; and FIG. 11F shows and array of
obstacles;
FIGS. 12A-12E schematically illustrate integration of an obstacle
array into a microfluidic fluidic device for emulsion generation;
FIG. 12A shows an obstacle array within a microfluidic channel;
FIG. 12B shows a multiplexing of arrays; FIG. 12C shows an example
of an on-chip amplification; FIG. 12D shows using membrane valves
and pumps to move liquid; FIG. 12E shows a closed version of a
membrane pump;
FIG. 13 schematically illustrates the breaking and deactivating of
an emulsion in two steps;
FIGS. 14A-14D schematically illustrate obstacle based bead sorting;
FIG. 14A shows unamplified beads appearing amplified; FIG. 14B
shows separation of amplified beads from unamplified beads by use
of obstacles; FIG. 14C shows a different implementation of the
separation approach; FIG. 14D shows a further implementation of the
separation approach;
FIG. 15 schematically illustrates integration of an enrichment
module into a microfluidic fluidic device;
FIGS. 16A-16D schematically illustrate emulsification using
obstacle arrays. FIG. 16A shows an obstacle array; FIG. 16B shows
another obstacle array; FIG. 16C shows a crude emulsion prior to
the fluid being directed through an obstacle array; FIG. 16D shows
an emulsion after the fluid is directed through an array;
FIG. 17 schematically illustrates systems for bead and bead free
nucleic acid sequencing;
FIG. 18 schematically illustrates thermodynamic movement of linear
polymer chains in solution;
FIG. 19 schematically illustrates an entangled polymer network
coupled to a solid support and primers;
FIG. 20 schematically illustrates a reaction between a
benzthiolated primer and Bromoacetamidepentylacrylamide
(BRAPA);
FIGS. 21A-21D schematically illustrate preparation of a porous
polymer monolith (PPM) thin film; FIG. 21A shows a support surface
to be modified; FIG. 21B shows the surface modification with a
polymerizable silane group; FIG. 21C shows the system before
polymerization; FIG. 21D shows the system after polymerization;
FIG. 22 schematically illustrates a polymerization chemistry for
PPM thin film preparation;
FIG. 23 schematically illustrates a hybridization-dehybridization
(Hyb-DeHyb) assay performed with a primer conjugated to the PPM
thin film;
FIG. 24 schematically illustrates procedure for preparing primer
conjugated polymer coated supports and visualization of primer
density;
FIG. 25 schematically illustrates primer density of primer
conjugated supports both with and without silane activation;
FIGS. 26A-26C schematically illustrate immobilized primer
arrangements with regard to the polymer coated support and method
of building confinement into the amplification reaction; FIG. 26A
schematically illustrates immobilization of primers to the polymer
coated support; FIG. 26B schematically illustrates a surface
confined amplification method; FIG. 26C shows template density
after amplification as a function of starting template
concentration;
FIG. 27 shows visualization of the primer density before and after
an amplification reaction;
FIG. 28 shows electronic detection of primer extension and nucleic
acid incorporation for templates amplified on a support
surface;
FIG. 29 shows capillary electrophoresis readout of extension of
FAM-labeled primers during a sequencing experiment;
FIGS. 30A and 30B show visualization of primer density and
electronic detection of nucleic acid incorporation on a support
surface; FIG. 30A shows visualization of primer density before and
after a amplification reaction; FIG. 30B shows visualization of
accumulated nucleic acid incorporation and electronic detection of
nucleic acid incorporation;
FIGS. 31A and 31B show the density of primers conjugated to polymer
coated supports before and after rinsing with an alkaline solution;
FIG. 31A shows primer density before rinsing with an alkaline
solution; FIG. 31B shows primer density after rinsing with an
alkaline solution;
FIG. 32 shows a thin-layer cell used for conjugation of primers to
a PPM thin film;
FIG. 33 shows detection of primer oligos surface conjugated to the
PPM thin film;
FIGS. 34A and 34B show an example clonal amplification method; FIG.
34A schematically depicts an example method for producing clonal
colonies of nucleic acid molecules; FIG. 34B schematically depicts
an example surface on which colonies of nucleic acids are present;
and
FIG. 35 schematically depicts an example method for sequencing a
nucleic acid molecule using redox mediators.
DETAILED DESCRIPTION
While various embodiments of the invention have been shown and
described herein, it will be obvious to those skilled in the art
that such embodiments are provided by way of example only. Numerous
variations, changes, and substitutions may occur to those skilled
in the art without departing from the invention. It should be
understood that various alternatives to the embodiments of the
invention described herein may be employed.
The term "adjacent to," as used herein, generally refers to next
to, in proximity to, or in sensing or electronic vicinity (or
proximity) of. For example, a first object adjacent to a second
object can be in contact with the second object, or may not be in
contact with the second object but may be in proximity to the
second object. An object adjacent to another object may have one or
more intervening objects (e.g., layers). In some examples, a first
object adjacent to a second object is within about 0 micrometers
(.mu.m), 0.001 .mu.m, 0.01 .mu.m, 0.1 .mu.m, 0.2 .mu.m, 0.3 .mu.m,
0.4 .mu.m, 0.5 .mu.m, 1 .mu.m, 2 .mu.m, 3 .mu.m, 4 .mu.m, 5 .mu.m,
10 .mu.m, or 100 .mu.m of the second object.
As used herein, the terms "amplifying", "amplification" and
"nucleic acid amplification" are used interchangeably and generally
refer to generating one or more copies or "amplified product" or
"amplicons" of a nucleic acid. Amplification of a nucleic acid may
be linear, exponential, or a combination thereof. Non-limiting
examples of nucleic acid amplification methods include reverse
transcription, primer extension, polymerase chain reaction, ligase
chain reaction, helicase-dependent amplification, asymmetric
amplification, rolling circle amplification, recombinase polymerase
amplification (RPA), and multiple displacement amplification (MDA).
In some embodiments, the amplified product may be DNA. In cases
where a target RNA is amplified, DNA can be obtained by reverse
transcription of the RNA and subsequent amplification of the DNA
can be used to generate an amplified DNA product. In cases where
DNA is amplified, DNA amplification may be employed. Non-limiting
examples of DNA amplification methods include polymerase chain
reaction (PCR), variants of PCR (e.g., real-time PCR,
allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR,
emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot
start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR,
multiplex PCR, nested PCR, overlap-extension PCR, thermal
asymmetric interlaced PCR, touchdown PCR), and ligase chain
reaction (LCR). In some cases, nucleic acid amplification is
dependent upon thermal cycling conditions. In other cases, nucleic
acid amplification is isothermal.
The term "bead," as used herein, generally refers to any type of
particle suitable for association with a nucleic acid or other
biological molecule. A bead may have a regular shape, including
spherical and non-spherical shapes and 1:1 aspect ratio and non 1:1
aspect ratios. In some cases, a bead has a regular shape (e.g., a
spherical bead) or may have an irregular shape (e.g., a
globular-bead comprising multiple domains of magnetic material). A
bead may comprise any type of suitable material(s) with
non-limiting examples that include metals, ceramics, magnetic
materials, a polymer(s) and combinations thereof. In some cases, a
bead is magnetic and, with a magnetic force applied to the bead,
can be manipulated/immobilized. Non-limiting examples of beads
include nanobeads (e.g., nanorods, nanospheres, nanoshells,
nanotubes, nucleic acid nanoballs, etc.), microbeads (e.g.,
microspheres, microbeads, etc.), quantum dots, cells, polymeric
scaffolds and combinations thereof.
The terms "nucleic acid," "nucleic acid molecule," "nucleic acid
fragment," "oligonucleotide" or "polynucleotide," are used herein
interchangeably, and generally refer to a molecule comprising one
or more nucleic acid subunits, or nucleotides. A nucleic acid may
include one or more nucleotides selected from adenosine (A),
cytosine (C), guanine (G), thymine (T) and uracil (U), or variants
thereof. A nucleotide generally includes a nucleoside and at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO.sub.3) groups.
A nucleotide can include a nucleobase, a five-carbon sugar (either
ribose or deoxyribose), and one or more phosphate groups.
Ribonucleotides are nucleotides in which the sugar is ribose.
Deoxyribonucleotides are nucleotides in which the sugar is
deoxyribose. A nucleotide can be a nucleoside monophosphate or a
nucleoside polyphosphate. A nucleotide can be a deoxyribonucleoside
polyphosphate, such as, e.g., a deoxyribonucleoside triphosphate
(dNTP), which can be selected from deoxyadenosine triphosphate
(dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine
triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine
triphosphate (dTTP) dNTPs, that include detectable tags, such as
luminescent tags or markers (e.g., fluorophores). A nucleotide can
include any subunit that can be incorporated into a growing nucleic
acid strand. Such subunit can be an A, C, G, T, or U, or any other
subunit that is specific to one or more complementary A, C, G, T or
U, or complementary to a purine (i.e., A or G, or variant thereof)
or a pyrimidine (i.e., C, T or U, or variant thereof). In some
examples, a nucleic acid is deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), or derivatives or variants thereof. A
nucleic acid may be single-stranded or double stranded. In some
cases, a nucleic acid molecule is circular. Moreover, a nucleic
acid can have any suitable length, such as a length of at least
about 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1
kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, or 50 kb.
Nucleic acids may include one or more non-standard nucleotide(s),
nucleotide analog(s) and/or modified nucleotides. Examples of such
nucleotides include, but are not limited to diaminopurine,
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-D46-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine,
aminoallyl-dUTP (aa-dUTP) and aminohexhylacrylamide-dCTP
(aha-dCTP). Nucleic acids may also be modified at a base moiety
(e.g., at one or more atoms that typically are available to form a
hydrogen bond with a complementary nucleotide and/or at one or more
atoms that are not typically capable of forming a hydrogen bond
with a complementary nucleotide), sugar moiety or phosphate
backbone.
The terms "nucleic acid sequencing" and "sequencing" are used
interchangeably and generally refer to the determination of a
nucleotide sequence of a nucleic acid. In some cases, the entire
nucleotide sequence of a nucleic acid is determined. In other
cases, only a portion of the nucleotide sequence of a nucleic acid
is determined. Nucleic acid sequencing can be conducted in any
suitable fashion, including via sequencing-by-synthesis. In
sequencing-by-synthesis, nucleotides are sequentially incorporated
into a primer hybridized to a template nucleic acid to-be-sequenced
(e.g., a growing nucleic acid strand), often via the action of an
enzyme such as a polymerase. During and/or after the incorporation
of each nucleotide, signals indicative of incorporation can be
detected. When such signals are associated with a particular type
of nucleotide (e.g., a nucleotide having an A, T, C, G or U base),
the signals can be used to determine the particular nucleotide
incorporated and, thus, via base-pairing rules, the complementary
nucleotide of the template nucleic acid to-be-sequenced. The
process of nucleotide incorporation and detection repeats until a
sequence of the nucleic acid is determined.
The term "polymerase," as used herein, generally refers to any
enzyme capable of catalyzing a polymerization reaction. A
polymerase can be naturally occurring or can be synthetic. In some
cases, a polymerase is a nucleic acid polymerase that is capable of
facilitating the sequential incorporation of nucleotides to a
primer hybridized (e.g., a growing nucleic acid strand) to a
template nucleic acid. Examples of nucleic acid polymerases include
a DNA polymerase, an RNA polymerase, a thermostable polymerase, a
wild-type polymerase, a modified polymerase, E. coli DNA polymerase
I, T7 DNA polymerase, bacteriophage T4 DNA polymerase .PHI.29
(phi29) DNA polymerase, Taq polymerase, Tth polymerase, Tli
polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase,
DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Sso
polymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4
polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma
polymerase, Tca polymerase, Tih polymerase, Tfi polymerase,
Platinum Taq polymerases, Tbr polymerase, Tfl polymerase, Tth
polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo
polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow
fragment, polymerase with 3' to 5' exonuclease activity, and
variants, modified products and derivatives thereof. In some
embodiments, a polymerase is a single subunit polymerase. Moreover,
a polymerase can have relatively high processivity, namely the
capability of the polymerase to consecutively incorporate
nucleotides to a primer without releasing the nucleic acid template
to which the primer is hybridized.
The term "nucleotide," as used herein, generally refers to an
organic molecule that serves as the monomer, or subunit, of a
nucleic acid molecule, such as a deoxyribonucleic (DNA) molecule or
ribonucleic acid (RNA) molecule. In some embodiments, a nucleotide
may also be a peptide nucleic acid (PNA) nucleotide or a locked
nucleic acid (LNA) nucleotide.
The term "primer," as used herein, generally refers to a strand of
nucleic acid that serves as a starting point for nucleic acid
synthesis, such as polymerase chain reaction (PCR). In an example,
during replication of a DNA sample, an enzyme that catalyzes
replication starts replication at the 3'-end of a primer attached
to the DNA sample and copies the opposite strand.
The term "clonal," as used herein, generally refers to at least
some, substantially all, or all, of the populations of a sensor
area being of the same nucleic acid sequence. There may be two
population associated with a single sample nucleic acid fragment,
as may be used for "mate pairs," "paired ends", or other similar
methodologies; the populations may be present in roughly similar
numbers in the sensor area, and may be randomly distributed over
the sensor area.
The term "confinement," as used herein, generally refers to a
species, moiety, or molecule generated (such as DNA) in one sensor
area staying associated with the same or substantially the same
sensor area so as to maintain or substantially maintain the clonal
nature of the sensor area.
The term "sample," as used herein, generally refers to a biological
sample. Examples of biological samples include nucleic acid
molecules, amino acids, polypeptides, proteins, carbohydrates,
fats, or viruses. In an example, a biological sample is a nucleic
acid sample including one or more nucleic acid molecules.
The term "reagent," as used herein, generally refers to one or more
substances that can be employed for use sample preparation or
analysis. Sample preparation can include sample processing. An
example of sample preparation is a nucleic acid amplification
reaction, such as polymerase chain reaction (PCR). Examples of
reagents for use with nucleic acid amplification reactions include
one or more primers and a polymerase, as well as cofactors (e.g.,
magnesium or manganese). In some cases, nucleic acid amplification
is not a PCR reaction.
The terms "surface modified" or "surface modification," as used
herein, generally refers to a surface in which the chemical or
physical (e.g., electronic) characteristic(s) has been changed or
altered. A chemical surface modification may include treating the
surface with an acid, base, or ozone. The chemically modified
surface may be coupled with a reactive linker. The reactive linker
may include, but is not limited to, silane coupling agents,
homobifunctional crosslinkers, heterobifunctional crosslinkers,
trifunctional crosslinkers, bifunctional chelating agents,
biotinylation reagents, or any other reactive functional group.
Physical surface modifications may include, but are not limited to,
wet etching and dry etching.
Systems for Detecting a Biomolecule
The present disclosure provides an integrated sequencing platform
that may include various components. The integrated sequencing
platform may be used in various applications, such as sequencing a
nucleic acid sample from a living subject.
In the context of nucleic acid sequencing, beads comprising clonal
populations of nucleic acid can be provided (e.g., via fluid flow,
perhaps through one or more fluid channels (e.g., microfluidic
channels) associated with the sensor array) to the sensor array,
and the beads immobilized to sensor sites. After bead
immobilization, reagents suitable for nucleic acid sequencing can
be serially contacted with the sensor array to sequence nucleic
acids on a bead associated with a given sensor site. For example,
in a first round, a sensor array may be contacted with a fluid
comprising a primer that can hybridize with nucleic acids and be
extended in template-directed fashion via the action of a
polymerase. The array can then be washed to remove any
non-hybridized primers. In a second round, the sensor array may be
contacted with a fluid comprising reagents suitable for primer
extension (e.g., polymerase, co-factors, suitable buffer) and a
particular known nucleotide (e.g., A, T, C, G or U). Incorporation
occurs and, at array sites where incorporation occurs, sensors at
those sites can detect signals indicative of incorporation via any
suitable modality, including one or more of those described
elsewhere herein. Where signal is detected, the signal can be
interpreted as incorporation of the particular known nucleotide
into the template. In some cases, the signal magnitude/intensity
can be used to determine how many of the particular known
nucleotides are incorporated into nucleic acids at a given site,
such as in the case where the known nucleotide is incorporated more
than once (e.g., in the case of a repeat template complement). The
sensor array can then be washed and the cycle repeated for each
remaining known nucleotide used for sequencing and the entire set
of cycles then repeated for each nucleic acid sample nucleotide,
until sample nucleic acids bound to beads are sequenced.
For example, a sensor array with sites occupied by beads comprising
clonal populations of nucleic acids can be contacted with a fluid
comprising a primer(s) that hybridize to clonal nucleic acids. The
sensor array can then be washed and then contacted with a fluid
comprising an adenine-containing nucleotide, a polymerase and any
necessary co-factors in a suitable buffer. Adenine-containing
nucleotide can incorporate to hybridized primers, where the next
template site on clonal nucleic acids is a thymine-containing
nucleotide. The array can then be washed and the
incorporate-and-wash cycle repeated for each of guanine-containing
nucleotides, thymine-containing nucleotides and cytosine-containing
nucleotides. Once the four types of nucleotides have been contacted
with the sensor array, that cycle can also repeat for another set
of contacting with each of the four types of nucleotides, until
sample nucleic acids bound to beads are sequenced.
In some cases, nucleic acid (e.g., deoxyribonucleic acid (DNA))
amplification and sequencing may be performed sequentially, or even
simultaneously, on the same array. In such cases, the array may
comprise components useful for one or both of amplification and
sensing at a given array location. In addition to having a feature
that can immobilize a bead, a given array site can include one or
more electrodes. The one or more electrodes may comprise one or
more separate electrodes that can provide an electric field that
generates a virtual well to confine reagents to an array site and
one or more separate electrodes that function as sensors. In some
cases, the one or more electrodes may comprise one or more
electrodes that can both provide an electric field and also
function as a sensor.
The sensor array may be incorporated into an integrated sequencing
platform. An integrated sequencing platform may include one or more
of a nucleic acid (e.g., DNA) extraction module, a library
construction module, an amplification module, an extraction module,
and a sequencing module. In some embodiments the systems may be
separate and/or in modular format. In some embodiments, the
integrated sequencing platform can include one, two, three, four,
or all five of these systems. In some cases, the modules can be
integrated within a single unit (e.g., a microfluidic device), a
single array (e.g., a sensor array that may be re-usable) or even a
single device. Examples of integrated sequencing platforms can be
found in PCT Patent Application No. PCT/US2011/054769, PCT Patent
Application No. PCT/US2012/039880, PCT Patent Application No.
PCT/US2012/067645, PCT Patent Application No. PCT/US2014/027544,
PCT Patent Application No. PCT/US2014/069624 and PCT Patent
Application No. PCT/US2015/020130, each of which is entirely
incorporated herein by reference.
An example of an integrated sequencing platform is schematically
depicted in FIG. 1. The integrated sequencing platform includes a
library construction module (e.g., nucleic acid library
construction system), which may include one or both of a nucleic
acid fragmentation element and a size selection element. As shown
in the example of FIG. 1, the library construction system includes
nucleic acid (e.g., DNA) fragmentation and size selection elements
in a single unit 116. Nucleic acid 112 provided to the
fragmentation and size selection unit 116 can be extracted from a
biological sample (e.g., a cell 110) and separated 120 from other
materials in the biological sample prior to fragmentation. The
fragmentation and size selection unit 116 can be configured to
produce nucleic acid fragments, such as double-stranded nucleic
acid fragments, which may or may not have blunted ends, via the
elements and methods described below. The fragmentation and size
selection unit 116 can include one or more microfluidic channels
122 within which nucleic acid may be disposed along with a set of
fragmentation beads 124. Nucleic acid 112 collected in a nucleic
acid (e.g., DNA) extraction system (shown for example in FIG. 1)
can be conveyed or "injected" into the nucleic acid (e.g., DNA)
fragmentation and size selection unit 116 by any suitable method
(e.g., pressurized injection, electrophoretic movement, gravity
feed, heat-induced movement, ultrasonic movement and/or the like).
Similarly, fragmentation beads 124 can be conveyed into the nucleic
acid (e.g., DNA) fragmentation element and size selection unit 116
by any suitable method.
The fragmentation element and/or size selection unit 116 can
include a pump 126 to produce movement of a fluid (e.g., a fluid
comprising nucleic acid (e.g., DNA) and fragmentation beads 124)
within a microfluidic channel 122. The pump 126 can be, for
example, a peristaltic pump, rotary pump, or reciprocating pump. In
some embodiments, the pump 126 can include one or more microfluidic
elements in fluid communication with the microfluidic channel 122,
and may have a flexible side-wall that, when deformed, produces a
flow within the microfluidic channel 122. In other embodiments,
however, another suitable strategy can be used as an alternative or
in addition to produce movement fluid within the microfluidic
channel 122, with non-limiting examples, that include selective
heating and cooling of the fluid, pneumatic pressurization of the
microfluidic channel, electrophoretic motion, or the like.
As shown in FIG. 1, The fragments 114 that are generated by the by
the size selection unit 116 can be transferred to an amplification
unit 132 along with beads 134 that are capable of binding the
fragments 114. The amplification unit 132 can include an array of
features that are each capable of retaining a bead. Beads may be
bound to the array via magnetic (e.g., via a magnetic feature),
electrostatic (e.g., via one or more electrodes), or via a member
of a binding pair (e.g., via hybridization of nucleic acid with
nucleic acid coupled to the array). In some cases, fragments may be
provided to an amplification unit at dilute concentrations in order
to obtain a desired ratio of molecules of sample nucleic acid to
beads. The flow rates of beads 134 and fragments 114 supplied to
the amplification unit 132 can be carefully controlled such that,
on average, less than one fragment is associated with a given bead.
Such a ratio can help to ensure the clonal nature of amplicon
populations generated in the amplification unit 132. Binding of the
fragments 114 to beads can be achieved via any suitable route,
including hybridization with an oligonucleotide coupled to the
beads, covalent linkages, an associated binding ligand pair, or any
other binding technique. The binding may be a non-reversible
covalent binding, a reversible covalent binding, or a reversible
non-covalent binding.
Reagents for amplification of bound fragments are provided to the
array and amplification of the fragments can then proceed in any
suitable fashion, including via a polymerase chain reaction (PCR),
strand-displacement amplification, isothermal amplification or any
other suitable amplification method, to generate a population of
beads, each bead comprising a clonal population of nucleic acids.
Nucleic acid amplification may be performed in multiple cycles.
Once a first round of amplification is completed after contacting
the array with a first set of nucleic acid fragments, the array may
be washed in order to remove any unbound amplicons and other
reagents in solution. Following washing, a second round of
amplification may be completed, by contacting the array with
additional nucleic acid fragments and then exposure of those
fragments to reagents suitable for nucleic acid amplification.
Where clonal amplification is complete (e.g., no binding sites
remain on some beads) the second fragments may bind only to beads
not already comprising amplicons, as sites with amplicons from
first round of amplification may be fully loaded with amplicons.
The process may be repeated for any number of amplification cycles
until capture sites are exhausted. Utilizing multiple rounds of
amplification may help eliminate double Poisson distribution
problems and help ensure that each sensor site is associated with
only one nucleic acid sequence, yet the occupancy of array sites is
maximized.
In some cases, a virtual well (e.g., generated via an electric
and/or magnetic field) produced by one or more electrodes at a
given array site can be implemented to confine amplification
reagents to the given array site. Virtual wells can permit
amplification of nucleic acids at a sensor position without
cross-contamination of reactants with those of other sensors of the
array. Amplification within a virtual well can aid in generating
clonal populations of amplicons.
Once amplification is complete, but prior to sequencing, clonal
beads generated from amplification can be transported to an
enrichment unit 138 that separates beads having amplicons from
those without nucleic acid. The enrichment module may include any
method of sorting beads comprising amplicons from those not
comprising amplicons. Sorting methods may include size selection,
electrophoretic sorting, or sorting by bead capture. Sorting bead
capture may include associating beads comprising amplicons with
capture moiety through hybridization, ligand pair binding, or any
other reversible binding technique. In some cases, separation
methods may make use of electrophoretic methods, implemented in an
electrophoretic sorter. In an electrophoretic sorter, null beads
(e.g., beads without nucleic acids), as well as beads subject to
incomplete amplification or those comprising overly short nucleic
acids, can be sorted from beads comprising desired amplicons via an
electrophoretic force (e.g., via an applied electric field)
generated by components of the sorter onto beads.
An electrophoretic sorter may comprise one or more channels capable
of accepting sorted beads. Beads with desired amplified product may
have sufficient charge to be directed to an outlet channel via
their interaction with an electric field. The sorted beads can be
collected from the outlet channel and provided back to the
amplification system for amplification. Moreover, beads without
appropriate amounts of amplified product and/or without amplicons
of adequate length may flow through the electrophoretic sorter and,
instead, be directed into a waste channel. The beads may be
collected from the waste channel and may be reused for another
cycle of amplification or other purpose upon appropriate cleaning
to remove any undesirable species. For example, beads may be washed
with a bleaching agent, such as hydrogen peroxide, to help ensure
that no contaminants remain on the beads so that they may be
reused.
Additional examples of enrichment systems and electrophoretic
sorters are described in PCT Patent Application No.
PCT/US2011/054769, PCT Patent Application No. PCT/US2012/039880,
PCT Patent Application No. PCT/US2012/067645, PCT Patent
Application No. PCT/US2014/027544, and PCT Patent Application No.
PCT/US2014/069624, each of which is entirely incorporated herein by
reference.
Once separation is complete, beads 140 that have amplicons can be
provided to a sequencing unit (e.g., a sequencing unit comprising a
sensor array 136) and sequenced. Enrichment of clonal beads may be
completed prior to sequencing. In some cases, enrichment of clonal
beads is not completed prior to sequencing. In such cases, the
amplified material generated during amplification is provided
directed to a sequencing unit and the nucleic acids bound to the
clonal beads are sequenced.
A sequencing unit may comprise one or more sensor arrays, each
array comprising a plurality of sites. A given site of the array
can include a sensor and, in some cases, a force mechanism for
immobilizing a bead at the given site and adjacent to its sensor.
In some cases, the force mechanism may be a magnet, such as, for
example, a permanent magnet or an electromagnet. A magnetic force
applied by the magnet can immobilize beads (e.g., beads comprising
a magnetic material) that are responsive to a magnetic force. In
some cases, the force mechanism comprises one or more elements
(e.g., one or more electrodes) that generate an electrostatic
force. An electrostatic force applied by the one or more elements
can immobilize beads (e.g., beads comprising a charged species,
such as nucleic acids) that are responsive to an electrostatic
force. In some cases, a given site of a sensor array comprises a
physical trench or well that can immobilize a bead. In some cases,
a given site of a sensor array comprises one or more molecules of
one member of a binding pair that can bind a bead comprising the
other member of the binding pair. Such members include an
oligonucleotide that can hybridize with another oligonucleotide
coupled to a bead, streptavidin or biotin that can bind with the
other of streptavidin and biotin coupled to a bead. Moreover, in
some cases, a sensing array may be free of wells and may be
substantially planar.
A sensor at a given position of a sensor array may be any suitable
sensor. In some cases, a sensor is an electronic sensor. An
electronic sensor can include one or more electrodes (in some
cases, at least two electrodes) that are capable of measuring
signals indicative of one or more of a change of impedance, a
change in charge, a change in ion concentration, and/or a change in
conductivity associated with a bead and/or a species coupled to a
bead. In some cases, a sensor may comprise NanoNeedle and/or
NanoBridge sensor and/or an optical sensor. A NanoBridge and/or
NanoNeedle sensors may be capable of detecting one or more of a
change in pH, a change in charge, a change in conductivity and a
change in impedance. NanoBridge and NanoNeedle sensors are
described in more detail in U.S. Patent Publication No. US
2012/0138460, PCT Patent Application No. PCT/US2011/054769, PCT
Patent Application No. PCT/US2012/039880, PCT Patent Application
No. PCT/US2012/067645, PCT Patent Application No.
PCT/US2014/027544, and PCT Patent Application No. PCT/US2014/069624
and PCT Patent Application No. PCT/US2015/020130, each of which is
entirely incorporated herein by reference.
Moreover, one or more sensors of a sensor array may be
independently addressable. An independently addressable sensor is
an individual sensor in an array whose response can be
independently detected from the responses of other sensors in the
array. An independently addressable sensor can also refer to an
individual sensor in an array that can be controlled independently
from other sensors in the array.
Sensing of a sensor may be based on one or more of local pH change,
local impedance change, local heat detection, local capacitance
change, local charge concentration (or change thereof), and local
conductivity change associated with a bead immobilized in proximity
to a sensor and/or species (e.g., nucleic acid) associated with the
bead. Changes in one or more of these measures can be effected by a
reaction (e.g., a nucleic acid sequencing reaction) involving a
species coupled to the bead and/or a binding event (e.g., nucleic
acid hybridization) involving a species coupled to the bead. Such
measurements can be made by directly detecting or detecting signals
that are indicative of a local pH change, local impedance change,
local heat detection, local capacitance change, local charge
concentration (or change thereof), and local conductivity change of
a bead or species (e.g., nucleic acid) coupled to a bead
immobilized in proximity to a sensor.
In some cases, one or more of these changes may be a change within
the Debye layer (or Debye length) of a bead or a species (e.g.,
nucleic acid) coupled to the bead or a sensor. The Debye layer may
have a characteristic thickness or length referred to as the Debye
length. Moreover, in some cases, sensing occurs within the Debye
layer (or Debye length) of (i) a bead (ii) a species associated
nucleic acid associated with the bead, or (iii) a sensor. A Debye
layer may be an electric (or electrical) double layer that is a
charge or conductivity boundary layer having a thickness around a
bead, species coupled the bead or a sensor, and/or the sensor. In
some cases, sensing occurs within the Debye layer spanning a sensor
and a bead. Furthermore, where a sensor comprises one or more
electrodes (e.g., at least two electrodes), the one or more
electrodes may be electrically coupled to the Debye layer of a bead
or a species coupled to a bead (e.g., a nucleic acid). In some
cases, the one or more electrodes may be within the Debye layer of
a bead or a species coupled to a bead (e.g., a nucleic acid). Such
sensor configurations are described, for example, in PCT Patent
Application No. PCT/US2011/054769, PCT Patent Application No.
PCT/US2012/039880, PCT Patent Application No. PCT/US2012/067645,
PCT Patent Application No. PCT/US2014/027544, and PCT Patent
Application No. PCT/US2014/069624, each of which is entirely
incorporated herein by reference.
Signals from the sensor may be detected transiently or during
steady state conditions. In a transient signal detection modality,
the detection occurs during or closely after a biological event,
such as nucleotide incorporation. In steady state detection,
reading of the sensor may occur after the "completion" of the
biological event or incorporation event. A steady state change in
signal may remain until a change is introduced to the environment
around the sensor. Steady state measurements may provide several
advantages over of the transient detection modality. The sensor may
be utilized in a manner whereby less data is requires as the sensor
may no longer be required to be read at a high data rate.
An example of a combined amplification and sequencing array and use
of the example array is depicted in FIGS. 2A-2F. As shown in FIG.
2A, the array 200 is configured on a substrate 201 (e.g., a
substantially planar substrate) that can comprise sensors (e.g.,
nanosensors) sometimes in communication with microfluidic channels
defined within the platform. Sensors may be associated with
substrate 201 and substrate 201 may also be associated with
magnetic 210 and electrode 205 and 207 elements. Magnetic beads may
be positioned over the sensors by the magnetic 210 or electrode 205
and 207 elements. The magnetic elements may form localized magnetic
fields and the electrode elements may form localized electric
fields in order to position a bead at various sensors of the array
200. Moreover, the magnetic and/or electric fields may create an
area of confinement for beads at occupied positions of the array
200.
As shown in FIG. 2B, a sample comprising nucleic acid 240 (e.g.,
nucleic acid fragments) may be conveyed to the array 200. Nucleic
acid 240 may be any suitable type of nucleic acid, including types
of nucleic acids described elsewhere herein. In some cases,
introduction of the nucleic acid 240 to the array 200 may be via
microfluidic channels associated with the array 200. As shown, the
array 200 may be configured with pre-localized magnetic beads 220
and the magnetic beads may be associated with primers capable of
hybridizing with nucleic acid 240, such that nucleic acid 240 is
captured by and becomes associated with the beads 220. The magnetic
beads 220 may be positioned on the array 200 via the magnetic
elements 210 and/or electrode 205 and 207 elements. Alternatively
or in addition, primers may be attached, bound, or associated with
a sensor at a position of the array 200 and used to trap nucleic
acid 240 at the sensor.
As shown in FIG. 2C, reagents 260 (e.g., polymerase,
deoxyribonucleotides (dNTPs), and additional primers) may be
simultaneously, previously, or subsequently introduced to the array
200. In some cases, introduction of the reagents 260 may be via
flow through microfluidic channels associated with the array 200,
such that the reagents 260 are contacted with the magnetic beads
220 via flow. Via magnetic and/or electrostatic forces from the
appropriate array elements, the magnetic beads 220 can be
maintained in the desired position as reagents 260 make contact
with the magnetic beads 220 via flow and/or during subsequent
amplification.
As shown in FIG. 2D, the nucleic acid 240 associated with magnetic
beads 220 can be clonally amplified to produce amplified nucleic
acid 245 on the surface of the magnetic beads 220. Clonal
amplification may be completed using any suitable method including
a method described herein such as, PCR, a primer extension
reaction, isothermal amplification, or other techniques.
As shown in FIG. 2E, the magnetic beads 220 on the array 200 may be
washed 280, removing unbound amplicons 247 and reagents 260 in
solution following amplification of nucleic acid 240. The result
can be magnetic beads 220 comprising clonal sets of amplified
nucleic acid 245 associated with array positions. Washing 280 may
be completed by any suitable method, such as, for example, washing
with a buffer solution at a flow rate sufficient to remove the
unbound amplicons 247 and reagents 260 in solution, but
insufficient to detach the magnetic beads 220 from their respective
positions on the array.
As shown in FIG. 2F, another aliquot of reagents 270 (e.g.,
polymerase, primers, etc.) and sequential cycles of individual
nucleotides 285 may then be contacted (e.g., via flow) with the
sensor array, permitting incorporation of the nucleotides into the
amplified nucleic acid 245 of magnetic beads 220. Nucleotides may
be introduced in individual cycles (e.g., cycle 1=A, cycle 2=T,
etc). There may be a wash step with buffer in between each cycle to
help reduce the chance of contamination from unincorporated
nucleotides. Polymerase used for the sequencing reaction, may be
the same type of polymerase that is used for the amplification
reaction, or may be a different type of polymerase, and can be
introduced prior to or with introduction of the nucleotides.
Detection of the incorporated nucleotides during each cycle can be
used to sequence the amplified nucleic acid 245, and, thus, the
original sample nucleic acid 240. Detection may occur, for example,
via one or both of electrodes 205 and 207. In some cases,
electrodes 205 and 207 can detect nucleotide incorporation events
by measuring local impedance changes of the magnetic beads 220
and/or the amplified nucleic acid (or other nucleic acid) 245
associated with the magnetic beads 220. Such measurement can be
made, for example, by directly measuring local impedance change or
measuring a signal that is indicative of local impedance change. In
some cases, detection of impedance occurs within the Debye length
(e.g., Debye layer) of the magnetic beads 220 and/or the amplified
nucleic acid 245 associated with the magnetic beads 220. Nucleotide
incorporation events may also be measured by directly measuring a
local charge change or local conductivity change or a signal that
is indicative of one or more of these as described elsewhere
herein. Detection of charge change or conductivity change can occur
within the Debye length (e.g., Debye layer) of the magnetic beads
220 and/or amplified nucleic acid 245 associated with the magnetic
beads 220.
Additional examples of combined amplification and sequencing
systems, for example, may be found in PCT Patent Application No.
PCT/US2011/054769, PCT Patent Application No. PCT/US2012/039880,
PCT Patent Application No. PCT/US2012/067645, PCT Patent
Application No. PCT/US2014/027544, and PCT Patent Application No.
PCT/US2014/069624, each of which is entirely incorporated herein by
reference.
Following the completion of sequencing, beads can be dissociated
from the array, the beads can be separated from bound species and
either or both of the beads and the array washed. Following
washing, the beads and array can be subsequently re-used for
another round of amplification and/or sequencing. Dissociation of a
bead from the array may be completed, for example, by
removal/reversal of a magnetic and/or electric field used to hold
the bead in place. In addition or as an alternative, fluid flow
and/or other type of field (e.g., external magnetic field, external
electric field) capable of exerting forces sufficient for
overcoming magnetic and/or electrostatic forces used to hold a bead
in place may also be used to dissociate the bead from an array.
While described herein with respect to nucleic acids and nucleic
acid sequencing reactions and nucleic acid hybridization, the
systems, devices, and methods described herein can be used for a
variety of other applications and detection of different biological
or biochemical moieties and reactions. Non-limiting examples of
such applications include antibody-antigen detection, protein or
peptide detection, protein or peptide binding/interaction
reactions, cell analysis, drug-discovery or screening, ligand
binding detection, pathogen detection, forensic analyses, small
molecule detection and reaction detection or other types of
analysis. Protein detection may be performed by direct measurement
of the reaction, by measurement of a sandwich assay, or by
measurement utilizing an aptamer. Sensing in the context of these
applications can be performed by coupling species or species that
participate in a to-be-monitored reaction to a bead and using
sensing methods described herein. In some cases, amplification
and/or sensing platforms can be useful in medical applications,
including point-of-care diagnostics.
The amplification and/or sensing platforms described herein can be
packaged into one or more devices. The device(s) can perform any
one or more of the operations of a method, including but not
limited to nucleic acid extraction, fragmentation, library
preparation, immobilization (e.g., on a bead), amplification,
confinement, bead enrichment, sequencing, or data analysis and
communication. In some cases, the device(s) are part of a detection
system. The system can include a single device of multiple devices.
Moreover, such a system can include a single module or may include
a plurality of modules. Each device can be for the same biological
detection or different biological detection. The devices can be in
communication with each other through any suitable type of
connectivity, including, for example, wireless connectivity.
An example device is shown in FIG. 3. FIG. 3 shows a detection
device 301, a removable chip 302 with a sensing array, and a
reagent reservoir 303 that can be inserted into and removed from
the biological detection device 301. The chip 302 can be a
single-use chip or multi-use chip. The chip can be disposable
(e.g., formed of an environmentally friendly material) and/or can
be reusable. Moreover, the sensing array can be configured as a
sensing array described herein and operated using a sensing method
described herein. In some cases, the sensing array also includes
electrodes for producing electric fields at sensor array positions.
In such cases, nucleic acid amplification and sensing can be
performed at a given location of the sensing array.
In some examples, the reagent reservoir 303 includes primers,
nucleotides and polymerase enzymes for nucleic acid sequencing. The
biological detection device 301 can include a screen 304 that can
include a user interface, such as a graphical user interface. The
screen 304 can enable a user to operate the device 301, such as for
nucleic acid sequencing. The biological detection device 301 can
include a port 305 that is configured to accept the removable chip
302. In some examples, upon insertion of the removable chip 302
into the device 301, nucleic acid sequencing can be performed using
the sensing array of the chip 302 and the reagents in the reagent
reservoir 303. In some cases, the device further comprises one or
more fluid flow paths in fluid communication with the sensing
array. The fluid flow path can also be in communication with one or
more reservoirs comprising one or more reagents for a biological
reaction (e.g., nucleic acid sequencing). In some cases, the fluid
flow path can provide beads to the sensing array in an emulsion or,
alternatively, without an emulsion.
In some situations, the device further comprises a computer
processor (or other electronic logic) coupled to the sensing array.
The computer processor can be programmed to receive signals from
the sensing array that are indicative of a direct electrical
signature of the species or reaction associated with the
species.
The device can be portable such that it can be readily transported
by a user or a machine. For example, the machine may be
transportable on a vehicle. In some examples, the vehicle is an
automobile, motorcycle, scooter, helicopter, airplane, truck,
military vehicle, spacecraft, or robot. In some cases, the sensing
device can be provided on a vehicle. The vehicle can be an
automobile, motorcycle, scooter, helicopter, airplane, truck,
military vehicle, spacecraft, or robot. The weight and footprint of
the device can be enclosed in a controlled to aid in rendering the
device portable. In some cases, the device may comprise a housing,
with a relatively small footprint, in which device components are
situated. Moreover, the device may be constructed of materials that
result in a relatively low weight of the device. In some examples,
the housing has a footprint that is less than or equal to about
250,000 (square millimeters) mm.sup.2, 200,000 mm.sup.2, 150,000
mm.sup.2, 100,000 mm.sup.2, 50,000 mm.sup.2, 10,000 mm.sup.2, 5,000
mm.sup.2, or 1,000 mm.sup.2 and the device has a weight that is
less than or equal to about 200 pounds, 175 pounds, 150 pounds, 125
pounds, 100 pounds, 75 pounds, 50 pounds, 25 pounds or 10
pounds.
The present disclosure provides computer control systems that are
programmed to implement methods of the disclosure. FIG. 4 shows a
computer system 401 that is programmed or otherwise configured to
operate device components, initiate and execute operation protocols
and/or process and analyze data obtained from sensing. The computer
system 401 can regulate various aspects of sensing devices, systems
and methods of the present disclosure, such as, for example,
methods for biological detection. In some embodiments, the computer
system 401 can receive signals from a sensor and determine a change
in local impedance, local charge and/or local conductivity as
described elsewhere herein.
The computer system 401 can be part of or separate from a device or
system for biological detection. In some examples, the system 401
is integrated with a device or system for biological detection,
such as a nucleic acid sequencing device. For example, the system
401 can be included in a housing that also contains a sensing
array, which can be provided via a removable chip.
The computer system 401 includes a central processing unit (CPU,
also "processor" and "computer processor" herein) 405, which can be
a single core or multi core processor, or a plurality of processors
for parallel processing. The computer system 401 also includes
memory or memory location 410 (e.g., random-access memory,
read-only memory, flash memory), electronic storage unit 415 (e.g.,
hard disk), communication interface 420 (e.g., network adapter) for
communicating with one or more other systems, and peripheral
devices 425, such as cache, other memory, data storage and/or
electronic display adapters. The memory 410, storage unit 415,
interface 420 and peripheral devices 425 are in communication with
the CPU 405 through a communication bus (solid lines), such as a
motherboard. The storage unit 415 can be a data storage unit (or
data repository) for storing data. The computer system 401 can be
operatively coupled to a computer network ("network") 430 with the
aid of the communication interface 420. The network 430 can be the
Internet, an internet and/or extranet, or an intranet and/or
extranet that is in communication with the Internet. The network
430 in some cases is a telecommunication and/or data network. The
network 430 can include one or more computer servers, which can
enable distributed computing, such as cloud computing. The network
430, in some cases with the aid of the computer system 401, can
implement a peer-to-peer network, which may enable devices coupled
to the computer system 401 to behave as a client or a server.
The CPU 405 can execute a sequence of machine-readable
instructions, which can be embodied in a program or software. The
instructions may be stored in a memory location, such as the memory
410. Examples of operations performed by the CPU 405 can include
fetch, decode, execute, and writeback.
The storage unit 415 can store files, such as drivers, libraries
and saved programs. The storage unit 415 can store user data, e.g.,
user preferences and user programs. The computer system 401 in some
cases can include one or more additional data storage units that
are external to the computer system 401, such as located on a
remote server that is in communication with the computer system 401
through an intranet or the Internet.
The computer system 401 can communicate with one or more remote
computer systems through the network 430. For instance, the
computer system 401 can communicate with a remote computer system
of a user (e.g., operator). Examples of remote computer systems
include personal computers (e.g., portable PC), slate or tablet
PC's (e.g., Apple.RTM. iPad, Samsung.RTM. Galaxy Tab), telephones,
Smart phones (e.g., Apple.RTM. iPhone, Android-enabled device,
Blackberry.RTM.), or personal digital assistants. The user can
access the computer system 401 via the network 430.
Methods as described herein can be implemented by way of machine
(e.g., computer processor) executable code stored on an electronic
storage location of the computer system 401, such as, for example,
on the memory 410 or electronic storage unit 415. The machine
executable or machine readable code can be provided in the form of
software. During use, the code can be executed by the processor
405. In some cases, the code can be retrieved from the storage unit
415 and stored on the memory 410 for ready access by the processor
405. In some situations, the electronic storage unit 415 can be
precluded, and machine-executable instructions are stored on memory
410.
The code can be pre-compiled and configured for use with a machine
have a processer adapted to execute the code, or can be compiled
during runtime. The code can be supplied in a programming language
that can be selected to enable the code to execute in a
pre-compiled or as-compiled fashion.
Aspects of the systems and methods provided herein, such as the
computer system 401, can be embodied in programming. Various
aspects of the technology may be thought of as "products" or
"articles of manufacture" typically in the form of machine (or
processor) executable code and/or associated data that is carried
on or embodied in a type of machine readable medium.
Machine-executable code can be stored on an electronic storage
unit, such memory (e.g., read-only memory, random-access memory,
flash memory) or a hard disk. "Storage" type media can include any
or all of the tangible memory of the computers, processors or the
like, or associated modules thereof, such as various semiconductor
memories, tape drives, disk drives and the like, which may provide
non-transitory storage at any time for the software programming.
All or portions of the software may at times be communicated
through the Internet or various other telecommunication networks.
Such communications, for example, may enable loading of the
software from one computer or processor into another, for example,
from a management server or host computer into the computer
platform of an application server. Thus, another type of media that
may bear the software elements includes optical, electrical and
electromagnetic waves, such as used across physical interfaces
between local devices, through wired and optical landline networks
and over various air-links. The physical elements that carry such
waves, such as wired or wireless links, optical links or the like,
also may be considered as media bearing the software. As used
herein, unless restricted to non-transitory, tangible "storage"
media, terms such as computer or machine "readable medium" refer to
any medium that participates in providing instructions to a
processor for execution.
Hence, a machine readable medium, such as computer-executable code,
may take many forms, including but not limited to, a tangible
storage medium, a carrier wave medium or physical transmission
medium. Non-volatile storage media include, for example, optical or
magnetic disks, such as any of the storage devices in any
computer(s) or the like, such as may be used to implement the
databases, etc. shown in the drawings. Volatile storage media
include dynamic memory, such as main memory of such a computer
platform. Tangible transmission media include coaxial cables;
copper wire and fiber optics, including the wires that comprise a
bus within a computer system. Carrier-wave transmission media may
take the form of electric or electromagnetic signals, or acoustic
or light waves such as those generated during radio frequency (RF)
and infrared (IR) data communications. Common forms of
computer-readable media therefore include for example: a floppy
disk, a flexible disk, hard disk, magnetic tape, any other magnetic
medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch
cards paper tape, any other physical storage medium with patterns
of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other
memory chip or cartridge, a carrier wave transporting data or
instructions, cables or links transporting such a carrier wave, or
any other medium from which a computer may read programming code
and/or data. Many of these forms of computer readable media may be
involved in carrying one or more sequences of one or more
instructions to a processor for execution.
The computer system 401 can include or be in communication with an
electronic display 435 that comprises a user interface (UI) for
providing, for example, an output or readout of device operation
and/or the signals obtained during and/or from sensing. Such
readout can include a nucleic acid sequencing readout, such as a
sequence of nucleic acid bases that comprise a given nucleic acid
sample. Examples of UI's include, without limitation, a graphical
user interface (GUI) and web-based user interface. The electronic
display 435 can be a computer monitor, or a capacitive or resistive
touchscreen.
Methods for Detecting Sensor Occupancy and Mitigating Motion
Artifacts
While immobilization of a bead associates the bead with a given
position of a sensor array, the bead can still move relative to the
electrodes, complicating the collection and analysis of a signal
sensed by a sensor. The present disclosure provides sensing methods
that can reduce, minimize and/or eliminate sensing complications
associated with bead movement at a sensor site. In general, such
methods utilize fluid conditions that can reduce, minimize and/or
eliminate the deleterious effects and inefficiencies of unoccupied
sensor sites, background noise, signal artifacts, and other signal
issues associated with bead movement.
The bulk flow of ions is relatively unaffected by the presence of
the bead at large distances away from the bead and is proportional
to the conductivity of the bulk fluid (.sigma..sub.b) in which
sensing takes place. However, at short distances, the electric
double layer (e.g., within the Debye length) has a surface current
that is proportional to the conductivity at the bead surface
(.sigma..sub.s). The relationship between these conductivities can
be represented by the Dukhin number (Du). The Dukhin number is a
dimensionless quantity defined as a ratio of the surface
conductivity (.sigma..sub.s) to the fluid bulk electrical
conductivity (.sigma..sub.b) multiplied by size of the bead (D):
Du=.sigma..sub.s/(.sigma..sub.bD). In the context of sensing, the
Dukhin number numerically represents the contribution of the
surface conductivity of the bead in overall current measured by the
sensor. Signal acquisition may be less sensitive to movements of
the bead when the conductivity of the bulk fluid is substantially
equal to the conductivity at the bead surface
(.sigma..sub.s.apprxeq.(.sigma..sub.b D), Du.apprxeq.1).
Alternatively, or in addition to, signal acquisition may be less
sensitive to movement of the bead when the conductivity of the bulk
fluid is slightly greater than the conductivity at the surface of
the bead (.sigma..sub.s<(.sigma..sub.bD), Du<1).
An aspect of the disclosure provides a method for sequencing a
nucleic acid template. The method comprises: (a) contacting a
nucleic acid template with a sensing fluid containing a population
of nucleotides, where the nucleic acid template is hybridized to a
primer and coupled to a bead, which bead is positioned proximate to
a sensor in a sensor array, where the sensor comprises a at least
two electrodes, and where the sensing fluid has a bulk conductivity
and a surface of the bead has a surface conductivity to provide a
Dukhin number that is less than about 1; (b) using the sensor to
detect a change in conductivity within a Debye layer of the bead
upon incorporation of at least one nucleotide of the population of
nucleotides into a growing nucleic acid strand, which growing
nucleic acid strand is derived from the primer and is complementary
to the nucleic acid template; (c) washing the sensor array to
remove unincorporated nucleotides of the population of nucleotides
from the sensor array; and (d) repeating (a)-(c) to obtain sequence
information for the nucleic acid template.
In some embodiments, the bulk conductivity is within about +/-500%,
within about +/-300%, within about +/-100%, within about +/-50% or
within about +/-10% of the conductivity of the surface of the bead.
In some embodiments, the Dukhin number determinable from the
conductivities of the sensing fluid and surface of the bead is less
than or equal to about 1, less than or equal to about 0.95, less
than or equal to about 0.9. less than or equal to about 0.8, less
than or equal to about 0.7, less than or equal to about 0.6, less
than or equal to about 0.5, less than or equal to about 0.4, less
than or equal to about 0.3, less than or equal to about 0.2, less
than or equal to about 0.1, or less. In some embodiments, the
Dukhin number determinable from the conductivities of the sensing
fluid and surface of the bead is greater than or equal to about
0.1, greater than or equal to about 0.2, greater than or equal to
about 0.3, greater than or equal to about 0.4, greater than or
about equal to 0.5, greater than or equal to about 0.6, greater
than or equal to about 0.7, greater than or equal to about 0.8,
greater than or about equal to about 0.9, greater than or about
equal to about 1, or greater. In some embodiments, the Dukhin
number determinable from the conductivities of the sensing fluid
and surface of the bead is between about 0.08 and 1, between about
0.1 and 1, between about 0.2 and 1, between about 0.3 and 1,
between about 0.4 and 1, between about 0.5 and 1, between about 0.6
and 1, between about 0.7 and 1, between about 0.8 and 1, or between
about 0.9 and 1. The sensing fluid may contain reaction components
necessary to sequence a nucleic acid template. The reaction
components may include one or more of polymerases or polymerizing
enzymes, buffers, salts, co-factors, adenosine triphosphate, and
crowding agents. The nucleotide may be incorporated with the aid of
the polymerase or polymerizing enzyme. A given electrode of a
sensor may be exposed to the sensing fluid. The electrodes may be
within a Debye layer of the bead or within a Debye layer of the
nucleic acid molecule to be sequenced. The sensor may further
detect changes in impedance within the Debye layer of the bead upon
incorporation of a nucleotide. The change in conductivity and the
change in impedance may be detected transiently or during steady
state conditions.
The sensing fluid may include one or more of magnesium chloride
(MgCl.sub.2) magnesium sulfate (MgSO.sub.4), sodium chloride
(NaCl), and potassium chloride (KCl), or any other buffer
comprising the desired conductivity. The sensing fluid may have a
solute concentration less than about 100 millimolar (mM), less than
about 50 mM, less than about 25 mM, less than about 15 mM, less
than about 10 mM, less than about 8 mM, less than about 6 mM, less
than about 4 mM, less than about 2 mM, less than about 1 mM, less
than about 0.8 mM, less than about 0.6 mM, less than about 0.4 mM,
less than about 0.2 mM, less than about 0.1 mM, or less. The
sensing fluid may have a solute concentration between about 0.1 mM
and 8 mM, between about 0.15 mM and 6 mM, between about 0.2 mM and
4 mM, between about 0.5 mM and 2 mM.
In another aspect, the disclosure provides a method for determining
bead occupancy at sites of a sensor array. The method comprises:
(a) contacting a sensor array with a plurality of beads, where the
sensor array comprises a plurality of sensors each having a at
least two electrodes, to provide a given bead of the plurality of
beads at a given position in proximity to an individual sensor of
the plurality of sensors; (b) contacting the sensor array with a
probe fluid that has a bulk conductivity that is at least about 50
times greater than or 50 times less than a conductivity associated
with a surface of the given bead; (c) using the individual sensor
to detect signals that are indicative of a presence of the given
bead in proximity to the sensor; and (d) identifying the given
position of the sensor array as occupied by the given bead.
The sensor array may be used to detect the presence of a bead
within sensing proximity of an individual sensor. Detecting a bead
within proximity of a sensor may include contacting the sensing
array with a probe fluid. The probe fluid may have a bulk
conductivity that is at least about 50 times greater than or 50
times less than the conductivity associated with the surface of the
bead. The sensor may be used to detect signals that are indicative
of a presence of the bead in proximity to the sensor. In some
embodiments, the conductivity associated with the probe fluid is at
least about 100 times greater, at least about 500 times greater, at
least about 1000 times greater, at least about 5000 times greater,
or more than the conductivity associated with the surface of the
bead. In some embodiments, the conductivity associated with the
probe fluid is at least about 100 times less, at least about 500
times less, at least about 1000 times less, at least about 5000
times less, or less than the conductivity associated with the
surface of the bead.
The probe fluid may have a bulk conductivity that is at least about
50 times great than or 50 times less than the conductivity of a
Debye layer of the given bead. The conductivity associated with the
probe fluid may be at least about 100 times greater, at least about
500 times greater, at least about 1000 times greater, at least
about 5000 times greater, or more than the conductivity of a Debye
layer of the given bead. The conductivity associated with the probe
fluid may be at least about 100 times less, at least about 500
times less, at least about 1000 times less, at least about 5000
times less, or less than the conductivity of a Debye layer of the
given bead.
The probe fluid can be any suitable buffer that has a conductivity
that is different or substantially different from the conductivity
at the bead surface, with non-limiting examples that include
magnesium chloride (MgCl.sub.2), magnesium sulfate (MgSO.sub.4),
sodium chloride (NaCl), potassium chloride (KCl) buffer, or any
other buffer. Moreover, the probe fluid can have any suitable
concentration of solutes, such that the conductivity of the fluid
is different or substantially different from the conductivity at
the bead surface. For example, the concentration of solutes in the
fluid may be from about 0.01 mM (millimolar) to about 1 molar (M),
0.01 mM to about 500 mM, 0.01 mM to about 50 mM, 0.01 mM to about
25 mM, 0.01 mM to about 10 mM, 0.1 mM to about 10 mM, 0.1 mM to
about 8 mM, 0.1 mM to about 6 mM, 0.1 to about 4 mM, 0.1 to about 2
mM or 0.1 to about 1 mM.
The Dukhin number may be determinable from the conductivity of the
probe fluid and the conductivity of the surface of the bead and may
be less than 1, greater than 1, substantially less than 1, or
substantially greater than 1. The bulk conductivity may be within
about +/-500% of a conductivity of the Debye layer. Contacting the
sensor array with a probe fluid may permit measurement of the
presence or absence of a bead in proximity to a sensor location.
The bead may be coupled to a nucleic acid molecule. Detecting the
presence of a bead in proximity to a sensor may be performed prior
to or during detection of the incorporation of nucleotides. Sensors
without a bead within sensing proximity may be excluded from
sensing and may not be used to detect a sequence of a nucleic acid
molecule. Alternatively, or in addition to, sensors with a bead
within sensing proximity may be used to detect a sequence of a
nucleic acid molecule. The signals to detect the presence of a bead
within sensing proximity to a sensor may comprise an electrical
current. The signals that comprise an electrical current may
include conductivity or impedance signals.
An example sensor and bead configuration is schematically depicted
in FIG. 5. With reference to FIG. 5, a bead 500 is located in
proximity to a first electrode 502 and a second electrode 504. The
bead 500 can move (in any direction) by a distance 504. Movement
can be due to various factors, including, for example, Brownian
motion, fluid flow, diffusion of vibration of the device, or a
combination thereof. In some cases, the bead 500 is continually
moving during detection of signal, complicating signal acquisition
and analysis with higher background noise and lower signal to noise
ratio.
The composition and concentration of species in a fluid (e.g.,
buffer) in which sensing takes place can affect the prominence of
sensing issues associated with bead movement. An example of the
charge environment in proximity is schematically depicted in FIG.
6. With reference to FIG. 6, a buffer comprising positive ions 600
and negative ions 605 can flow and/or conduct current in proximity
to a bead 610. Signal acquisition can be less sensitive to
movements of the bead when the conductivity of the bulk fluid is
substantially equal or equal to the conductivity at the bead
surface (.sigma..sub.s= or .apprxeq.(.sigma..sub.b D), Du= or
.apprxeq.1). For example, the conductivity of the fluid in which
sensing takes places may be within about plus or minus (+/-) 500%,
within about +/-450%, within about +/-400%, within about +/-350%,
within about +/-300%, within about +/-250%, within about +/-200%,
within about +/-150%, within about +/-100%, within about +/-50%,
within about +/-40%, within about +/-30%, within about +/-20%,
within about +/-10%, within about +/-5%, or within about +/-1%, of
the conductivity at the bead surface. In some examples, the
conductivity of the bead at its surface is within about +/-300%,
within about +/-250%, within about +/-200%, within about +/-150%,
within about +/-125%, within about +/-100%, within about +/-95%,
within about +/-90%, within about +/-85%, within about +/-80%,
within about +/-75%, within about +/-70%, within about +/-65%,
within about +/-60%, within about +/-55%, within about +/-50%,
within about +/-45%, within about +/-40%, within about +/-35%,
within about +/-30%, or within about +/-25% of the conductivity of
the fluid.
The fluid in which sensing occurs can be any suitable buffer that
has a conductivity that is substantially equal to or equal to the
conductivity at the bead surface, with non-limiting examples that
include magnesium chloride (MgCl.sub.2), magnesium sulfate
(MgSO.sub.4), sodium chloride (NaCl) and potassium chloride (KCl)
buffer. Moreover, the fluid in which sensing occurs can have any
suitable concentration of solutes, such that the conductivity of
the fluid is equal to or substantially equal to the conductivity at
the bead surface. For example, the concentration of solutes in the
fluid may be from about 0.01 mM (millimolar) to about 1 molar (M),
01 mM to about 500 mM, 0.01 mM to about 50 mM, 0.01 mM to about 25
mM, 0.01 mM to about 10 mM, 0.1 mM to about 10 mM, 0.1 mM to about
8 mM, 0.1 mM to about 6 mM, 0.1 to about 4 mM, 0.1 to about 2 mM or
0.1 to about 1 mM.
Data obtained from example sensing experiments evaluating the
effect of Du on observed signal with an electrode pair is
graphically depicted in FIG. 7. With reference to FIG. 7, the
vertical axis is dimensionless current (I.sub.r/I.sub.b) and the
horizontal axis is a Du, with a relatively high concentration
buffer represented to the left of the horizontal axis (e.g., toward
10.sup.-3) and a relatively low concentration buffer to the right
(e.g., toward 10.sup.3) of the horizontal axis. Each line on the
chart represents a different distance by which a bead has moved
relative to the sensor (ax) and ranges from 0 to about 0.8
micrometers (.mu.m). Note that in the region where the
conductivities are between Du=0.1 and Du.apprxeq.1 (i.e.,
approximately equal), sensing is least sensitive to bead movement
(e.g., all values of .DELTA.x give approximately the same current
signal). In other words, the sensor does not detect a significant
change in current when the bead moves relative to the sensor
electrodes. Conversely, also shown in FIG. 7, at high Du numbers,
the signal change 705 is relatively large. Such difference in
signal may be used to detect beads at sensor positions as described
elsewhere herein.
Additionally, differences in bulk conductivity of a fluid in which
sensing takes place and the conductivity of a surface of a bead can
be exploited to identify sensor array sites that are occupied with
a bead. Exposure of a sensor array to a probe fluid for which Du is
> or >>1 or Du< or <<1 can result in relatively
large signal changes that can be detected and interpreted to
indicate the presence of a bead at a given sensor. Identification
of sensors occupied with beads can reduce the amount of data
collected, by excluding data acquisition from sensors that are not
associated with a bead (and, thus, an analyte). Reduced data
acquisition can improve the speed and decrease the complexity of
data processing. Moreover, such differences can be used to
determine if a bead has been lost or gained from a particular
sensor location during or after sensing. Indeed, a sensor can be
exposed to a fluid for which Du> or >>1 or Du< or
<<1 at any point before or after sensing in order to
determine the bead occupancy of sensor array sites. For sensing,
the fluid can be replaced with one in which Du= or 1 or
0.1<Du.ltoreq.1 in order to minimize signal acquisition issues
as is described elsewhere herein.
Where a probe fluid is used to evaluated bead occupancy at various
sites of a sensor array, the conductivity at a bead surface can be
any conductivity sufficiently different (e.g., greater or less)
from the conductivity of the probe fluid such that an observable
signal change is generated at bead-occupied sensor sites. In some
cases, the conductivity of probe fluid is at least about 10 times,
at least about 50 times, at least about 100 times, at least about
500 times, at least about 1000 times, at least about 5000 times, at
least about 10000 times, at least about 50000 times, or at least
about 100000 time greater than the conductivity of the bead
surface. In some instances, the conductivity of the probe fluid is
at least about 10 times, at least about 50 times, at least about
100 times, at least about 500 times, at least about 1000 times, at
least about 5000 times, at least about 10000 times, at least about
50000 times, or at least about 100000 time less than the
conductivity of the bead surface.
Moreover, as stated previously, the Du determinable from the
conductivities of the probe fluid and surface of the bead can be
any suitable number, such that that Du is sufficiently greater or
less than 1 such that a sufficient signal change can be observed.
For example, the Du determinable from the conductivities of the
probe fluid and surface of the bead may be at least about 0.0001,
at least about 0.001, at least about 0.01, at least about 0.1, at
least about 1, at least about 10, at least about 100, at least
about 1000 or at least about 10000 or higher. In other examples,
the Du determinable from the conductivities of the probe fluid a
surface of the bead may be less than about 10000, less than about
1000, less than about 100, less than about 10, less than about 1,
less than about 0.1, less than about 0.01, less than about 0.001 or
less. In some examples, the Du determinable from the conductivities
of the probe fluid and surface of the bead may be from about 0.001
to about 0.9, from about 0.01 to about 0.9, from about 1.1 to about
10000 from about 10 to about 10000, or from about 1.1 to about
1000, from about 10 to about 1000, or from about 100 to about
1000.
The probe fluid can be any suitable buffer that has a conductivity
that is different or substantially different from the conductivity
at the bead surface, with non-limiting examples that include
magnesium chloride (MgCl.sub.2), magnesium sulfate (MgSO.sub.4),
sodium chloride (NaCl) and potassium chloride (KCl) buffer.
Moreover, the probe fluid can have any suitable concentration of
solutes, such that the conductivity of the fluid is different or
substantially different from the conductivity at the bead surface.
For example, the concentration of solutes in the fluid may be from
about 0.01 mM (millimolar) to about 1 molar (M), 0.01 mM to about
500 mM, 0.01 mM to about 50 mM, 0.01 mM to about 25 mM, 0.01 mM to
about 10 mM, 0.1 mM to about 10 mM, 0.1 mM to about 8 mM, 0.1 mM to
about 6 mM, 0.1 to about 4 mM, 0.1 to about 2 mM or 0.1 to about 1
mM.
Experimental data obtained from an example sensor occupancy
experiment is shown in FIG. 8. FIG. 8 shows an image 800 of a
30.times.30 sensor array, with various sensor sites occupied with
beads (white circles of 800). FIG. 8 also shows a signal output map
805 generated by exposing the sensor array to a probe fluid having
sufficient conductivity difference from the conductivity at a bead
surface bead such that a signal change (e.g., black areas of 805)
is observed at occupied sites. Conversely, no signal change (e.g.,
white areas of 805) is observed in areas unoccupied by a bead.
Correspondence between visually occupied and non-occupied sites in
photograph 800 and sites indicated as occupied on output map 805 is
good, validating the method.
In another example, the feasibility of discriminating bead-occupied
sensors from unoccupied sensors in a probe fluid (e.g., Du> or
>>1 or Du< or <<1) and sensing fluid (e.g., Du= or
.apprxeq.1) were evaluated in an example experiment exposing a
sensor array to each fluid. The results of the experiment are
graphically depicted in FIG. 9. As shown in FIG. 9, the measured
signal in the probe fluid distinguished occupied sites from those
unoccupied, indicated by a separation of signal in plot 905 of FIG.
9. Conversely, measured signal in the sensing fluid was not able to
distinguish occupied sensor sites from those unoccupied, indicated
by a lack of signal separation in plot 900 of FIG. 9.
Methods for Processing Nucleic Acid Samples
In an aspect, the present disclosure provides a method for
processing a nucleic acid sample. The method may comprise providing
a mixture comprising a first set of droplets and a second set of
droplets. The first droplet of the first set of droplets may
comprise (i) a bead, (ii) a recombinase, (iii) a polymerizing
enzyme, and (iv) a nucleic acid molecule from the nucleic acid
sample. The second droplet of the second set of droplets may
comprise an activating agent that increases a rate at which the
recombinase processes the nucleic acid molecule to permit the
primer to hybridize to the nucleic acid molecule to conduct a
primer extension reaction in the presence of the polymerizing
enzyme, to generate an amplification product(s) from the nucleic
acid molecule.
The first droplet may be merged with the second droplet in the
mixture to generate a third droplet as part of a third set of
droplets. The third droplet may comprise the bead having coupled
thereto the nucleic acid molecule, recombinase, primer and
polymerizing enzyme.
The primer extension reaction may be conducted to generate the
amplification product(s) from the nucleic acid molecule in the
third droplet. For example, the primer extension reaction may
include conducting isothermal recombinase polymerase amplification
(RPA) or a polymerase extension reaction (PCR).
FIG. 10 schematically illustrates an example of a method for
processing a nucleic acid sample. A mixture comprising a first set
of droplets and a second set of droplets may be merged to form a
third set of droplets. Formation of the third set of droplets may
activate the primer extension reaction within the confinement of
the third droplet.
The mixture comprising the first and second set of droplets may
further comprise an oil phase, such as mineral oil, and an
emulsifier, such as Tegosoft DEC or ABIL WE09.
The droplets may contain additional components. For example, the
first set of droplets may contain buffer, salts, crowding agents,
dNTPs, and primers. The second set of droplets, along with
containing buffer, salts, crowding agents, dNTPs, and primers, may
also contain ATP, recombinase loading enzyme, single-stranded
DNA-binding protein, and an ATP-regenerating unit (e.g.,
ATP-regenerating compound).
In some embodiments, the activating agent contained within the
second droplet may be a magnesium salt. Example magnesium salts
include, but are not limited to, magnesium chloride and magnesium
acetate.
Formation of the third droplet may be achieved quickly, on the
order of at most about 10 minutes, at most about 5 minutes, at most
about 1 minute, at most about 30 seconds, at most about 10 seconds,
at most about 5 seconds, or less, through low speed agitation. This
agitation may include stirring and/or shaking.
In some cases, the third set of droplets is directed through a set
of obstacles to control the shape or size of each of the third set
of droplets. The obstacles may be pillars, for example, and may be
comprised of a variety of shapes and sizes arranged in a variety of
structural patterns. The shape of the obstacles may include
triangles, squares, diamonds, and/or circles. The structural
patterns may include such arrangements as comb-like structures. The
obstacles and structural pattern may be formed on an elastic or
inelastic substrate. The obstacles and structural pattern may be
formed by photolithography, stamping, etching, machining, molding,
or any other fabrication approach. The size and shape of the third
set of droplets may further be controlled by the flowrate and
pressure used to direct the droplets through the set of obstacles.
The flowrate and pressure may be controlled by a pressurizing
reservoir or a membrane pump. Examples of obstacles and uses of
such obstacles are schematically illustrated in FIGS. 11A-11F. FIG.
11A illustrates the aqueous phase 1101 in proximity to an obstacle
1104. The aqueous phase 1101 may contain primer bound beads 1102
and may be surrounded by a continuous oil phase 1103. Flow may
transport the aqueous phase droplet towards the obstacle, which may
cause the droplet to deform and elongate 1105 due to shear forces,
as shown in FIG. 11B. FIG. 11C shows the droplet after it has
passed an example obstacle and split into two daughter droplets
1106A and 1106B. The daughter droplets 1106A and 1106B may each
contain a single primer bound bead 1102. The obstacles may have any
arbitrary shape, as seen in FIG. 11D, such as round 1104, square
1105, diamond 1106, and triangle 1107. Furthermore, obstacles may
be placed into a flow cell, see FIG. 11E. The flow cell may be
formed by two sidewalls 1107A and 1107B and the obstacles may form
a structural array 1112 pattern between the sidewalls 1107A and
1107B. The structural array, shown in FIG. 11F, may be defined by
the spacing and number of obstacles in a row 1108. Additional rows
may be defined by column offset 1109 and row offset 1110. The
boundary of the array may also contain partial obstacles 1111.
The flow cell containing the obstacle array may further be
integrated into a microfluidic fluidic device for emulsion
generation. Examples of the integration of an obstacle array into a
microfluidic fluidic device for emulsion generation is
schematically illustrated in FIGS. 12A-12E. FIG. 12A shows the
obstacle array 1112 placed into the microfluidic emulsion generator
device 1201 with an inlet channel 1203 and an outlet channel 1204.
FIG. 12B shows two multiplexed arrays. Multiplexing of arrays may
be implemented with, for example, a single inlet channel 1203, two
arrays 1112A and 1112B, and outlet channels 1204A and 1204B. FIG.
12C shows an example device for on-chip amplification. An on-chip
amplification reaction may be performed by connecting reagent
chambers, mixer, emulsion generator, and incubator by channels
carrying the fluids 1205. Transport of fluidics may be achieved by
an external pressure source and pressure lines 1206 connected to
reagent compartments as well as valves 1207. Valves and pressure
may be controlled by a control unit. Alternatively to pressurizing
reagent chambers, on-chip membrane valves and pumps, shown in FIGS.
12D and 12E, may be used to move liquid. The closed version of the
membrane pump 1208A may consist of a pneumatic layer 1209 with a
vacuum and/or pressure connector 1212, a membrane 1211A, and a
hydraulic layer 1210A and 1210B. Fluid may enter the pump using an
inlet 1213 and may exit the pump via an outlet 1214. The membrane
pump 1208B may be opened by applying vacuum to the pneumatic layer
to deform the membrane 1211B and liquid may enter the pump
compartment. Applying pressure to the pneumatic layer may close the
pump and liquid may flow out of the pump compartment. Surrounding
valves 1207, which may be made out of a small version of the
membrane pump, may direct the flow of liquid.
The third set of droplets may be disrupted. The result of
disrupting the third set of droplets may be the formation of a
homogenous solution. Disruption may be performed through the
addition of a disrupting mixture. As an alternative, this may be
performed by agitating the emulsion. The third set of droplets may
be disrupted after the primer extension reaction has been
conducted. An example of the breaking of an emulsion is
schematically illustrated in FIG. 13.
In some cases, the disrupting mixture may be comprised of a
deactivating agent dissolved in an emulsion disruptor. A variety of
emulsion disrupters may be used including polyethylene glycol like
compounds, such as triethylene glycol. A variety of deactivating
agents may also be used, such as tetraalkyl ammonium dodecylsulfate
compounds. An example of an effective deactivating agent is
tetrabutylammonium dodecylsulfate.
In some embodiments, beads coupled to amplification product(s) may
be captured by a capture bead to form a multi-bead complex. The
multi-bead complex may be appreciably larger than a non-complexed
bead. The multi-bead complex may be about 2 time larger, about 4
times larger, about 6 times larger, about 8 times larger, about 10
times larger, about 15 times larger, about 25 times larger, about
50 times larger, about 100 times larger, or more than the
non-complexed bead. The capture bead used may be chosen such that
it exclusively binds to beads coupled to amplification product(s).
The capture bead may include any moieties that reversibly bind to
the beads, such as through association/disassociations interactions
or hybridization events. Formation of a multi-bead complex may
enable separation and enrichment of beads coupled to amplification
product(s). Enrichment may be performed by size selection.
Alternatively, magnetic capture beads may be used to enable
enrichment to be performed using a magnet.
In some cases, size selection may be performed by directing beads
through a set of obstacles, wherein the non-complexed beads can
pass through the set of obstacles and the multi-bead complex
cannot. The obstacles may be pillars, for example, and may be
comprised of a variety of shapes and sizes arranged in a variety of
structural patterns. The size selection cutoff may be controlled by
obstacle shape, spacing, or structure. The shape of the obstacles
may include triangles, squares, diamonds, and/or circles. The
structural patterns may include such arrangements as comb-like
structures. The size selection may further be controlled by the
obstacle shape, the obstacle spacing, and/or the obstacle
structure.
Obstacles may be used to sort beads. Examples of obstacle based
bead sorting are schematically illustrated in FIGS. 14A-14D. Due to
the Poisson distribution, a subset of beads may be coupled to
amplification product(s) 1401 while a large portion of beads may
not be coupled to amplification product(s) 1402. Amplified beads
may be tagged exclusively and may be captured by larger capture
beads 1403, forming multi-bead complexes 1404, as shown in FIG.
14A. Separation of the multi-bead complexes from the non-complexed
beads may be achieved using a microfluidic enrichment module
containing an obstacle array 1405. An example enrichment module is
shown in FIG. 14B. A mixture a beads may enter the module through
an inlet channel 1406A. The Obstacle array 1104 may create a
filter-like structure, enabling the non-complexed beads to pass,
while the large multi-bead complex may be rejected. The channel
outlet 1408 may show an enrichment of multi-bead complexes and the
channel outlet 1407A may exclusively contain non-complexed beads.
The enriched portion may be recycled to inlet 1406A. Alternatively,
a different implementation, shown in FIG. 14C, may be used that
comprises a sole exit channel 1407B. In this implementation, the
captured multi-bead complexes may remain in the obstacle entry
channel 1406B and elusion may require reversal of the flow. A
further implementation of this concept, shown in FIG. 14D, may
contain a sheath flow 1411 to increase the effectiveness of the
enrichment.
An example of integration of the enrichment module 1512 integrated
into a microfluidic device is schematically illustrated in FIG. 15.
The sheath flow based enrichment module maybe connected to four
membrane pumps 1208, which may in turn be connected to valves.
Reservoir 1 may contain a bead mixture comprising multi-bead
complexes and non-complexed beads. Reservoir 3 may contain a
buffered aqueous solution. The membrane pumps may enable flow from
reservoir 1 and 3 through the enrichment module. Reservoir 2 may
collect the fraction of non-complexed beads after the bead mixture
has passed through the enrichment module. The portion of the bead
mixture containing the multi-bead complexes may be recycled to
reservoir 1 via a connector channel 1501, enabling additional
passes through the enrichment module. Additional passes through the
enrichment module may increase the enrichment levels to achieve a
sufficiently high ration of multi-bead complexes to non-complexed
beads.
A crude emulsion that has been directed through an array of
obstacles is shown in FIGS. 16A-16D. FIGS. 16A and 16B show example
arrays of round obstacles arranged in a comb-like or hexagonally
close packed arrangement. FIG. 16C shows a crude emulsion prior to
being directed through an array of obstacles. The crude emulsion
has droplet sizes that are large and non-uniform. The large
droplets may be prone to interaction, recombining, and forming
larger droplets. FIG. 16D shows an emulsion after being directed
through an array of obstacles. The droplet size in after being
directed through the array of obstacles are smaller and more
uniform than the crude emulsion. The smaller, more uniform droplets
may be more stable than the droplets of the crude emulsion.
Methods for Bead-Free Nucleic Acid Sequencing
The present disclosure provides methods and systems for bead-free
nucleic acid sequencing. This may involve using nucleic acid
amplification reaction, such as invader amplification, as described
in, for example, U.S. Patent Publication No. 2015/0344943, each of
which is entirely incorporated herein by reference.
In an aspect, the present disclosure provides a method for
processing a nucleic acid sample. The method may comprise
activating a sensor comprising a support that may comprise at least
two electrodes and a polymeric material adjacent to the support.
The electrodes may be exposed to a solution comprising the
polymeric material. The polymeric material may retain the nucleic
acid molecules during a sequencing reaction. The sequencing
reaction may yield signals indicative of individual bases of the
nucleic acid molecule. During the sequencing reaction, the
electrodes of the sensor may be used to detect the signals
indicative of individual bases of the nucleic acid molecule.
Signals may be measured transiently or during steady state
conditions.
The method may comprise performing a sequencing reaction which may
yield signals indicative of impedance changes that may accompany
the incorporation of nucleotides into the nucleic acid molecule.
During the sequencing reaction, the electrodes of the sensor may be
used to detect impedance signals indicative of nucleotide
incorporation into the nucleic acid molecule.
In another aspect, the present disclosure provides a system for
processing a nucleic acid sample. The system may comprise a support
comprising at least two electrodes and a polymeric material
adjacent to the support. The electrodes may be exposed to a
solution comprising the polymeric material and the polymeric
material may retain the nucleic acid molecule during a sequencing
reaction. The sequencing reaction may yield signals indicative of
individual bases of the nucleic acid molecule. The sensor may be
coupled to one or more computer processors that may operate the
sensor. The computer processors may be programmed to subject the
nucleic acid molecule to the sequencing reaction and to use the
electrodes of the sensor to detect signals indicative of individual
bases of the nucleic acid molecule. The computer processor may also
be programmed to use the detected signals to generate a sequence of
the nucleic acid molecule. The system may be configured to measure
transient signals, steady state signals, or both transient and
steady state signals.
The system may comprise a sequencing reaction which may yield
signals indicative of impedance changes accompanying incorporation
of nucleotides into the nucleic acid molecule. The sensor may be
coupled to one or more computer processors that operate the sensor
and may be programmed to use the electrodes to detect signals
indicative of impedance changes. The computer processor may also be
programmed to use the impedance changes to generate a sequence of
the nucleic acid molecule.
The sensor may be used to detect a variety of biological events.
Biological events may include, but are not limited to, DNA
sequencing, capture and detection of pathogens, protein and
biomarker detection, and other molecular diagnostics.
FIG. 17 shows an example system for both a bead-based sequencing
approach and a bead free sequencing approach. In this example, the
conjugated biomolecules are nucleic acid molecules. In the bead
approach, there are a limited amount of nucleic acid molecules at
the contact point of the two electrodes, which can result in a
non-optimal signal strength. Additionally, due to the
polydispersity of the particle size, the magnetic bead in this
illustration may slide laterally. Sliding laterally may result in
the bead losing contact with the electrode, creating signal noise.
Sliding laterally may also cause a no-close circuit which may cause
a loss of signal. By controlling the molecular weight, a physically
entangled polymeric material may bridge the two electrodes or cover
the electrodes, maximizing the detection of biological events.
An additional drawback to the bead-based approach may be the
potential for thermodynamic movement of linear polymer chains in
solution. FIG. 18 shows an example of a magnetic bead with a
polymer linker attached to a fifteen base primer. The linker,
comprising a poly(ethylene oxide) chain, is eighty-five atoms in
length. If fully extended, the conductivity of the region in the
vicinity of the bead can be similar to that of the conductivity of
the bulk. However, when the 85-atom linker collapses, it may create
a restively domain that may contribute to the background noise,
alter the linearity, and affect other unknown signal parameters. A
physically entangled polymeric material may mitigate such extending
and collapsing phenomenon.
The electrode may be formed of a chemically inert conducting
material, such as one or more metals (e.g., platinum, gold, or
titanium). The electrode may be formed of a conductive metal oxide,
for example, but not limited to, indium tin oxide (ITO). The
support may comprise an oxide (e.g., a silicon oxide), such as
silicon dioxide or a metal oxide. The support may be surface
modified to enable the reactive coupling of polymeric materials to
the surface of the support. Surface modification may include
chemical modification by treatment with acids, bases,
Ultraviolet-ozone treatement, or plasma treatment. Surface
modification may allow for the reactive coupling of a linker
molecule. Linker molecules may include crosslinkers or coupling
agents. Non-limiting examples of crosslinkers include zero-length
crosslinkers, homobifunctional crosslinkers, heterobifunctional
crosslinkers, and trifunctional crosslinkers. Non-limiting examples
of coupling agents include biotinylation reagents and silane
coupling agents. For example, the surface may be modified with an
amino-alkyl alkoxysilane. The amino-alkyl alkoxysilane may act as a
coupling agent that couples the polymeric material to the support.
The amino-alkyl alkoxysilane may contain one to three alkoxy groups
that may react with hydroxyl groups on the surface of the
substrate. The amino functional group of the amino-alkyl
alkoxysilane may be replaced with other functional groups and/or
may also contain a secondary reactive component (e.g., functional
group) that may couple with the polymeric material. Example
functional groups may include acrylamide, acrylate, thiol,
maleimide, carboxylate, NHS ester, tetrafluorophenyl ester,
pentafluorophenyl ester, epoxide, biotin, or streptavidin moieties.
The polymeric material may bridge or cover the electrodes.
The polymeric material may be a gel, such as a hydrogel. The
hydrogel may comprise non-reactive and reactive co-monomers. The
hydrogel may contain one or more of acrylate, methacrylate,
ethylene glycol, acrylamide, epoxide, vinyl alcohol, or
(hydroxyethyl)methacrylate monomers. The hydrogel may comprise
reactive copolymers, that may, for example, comprise a
water-soluble non-reactive co-monomer, with non-limiting examples
of such monomers including, 2-hydroxyethyla acrylate, vinylmethyl
ether, acrylamide and N,N-dimethylacrylamide. In some cases, the
hydrogel may also comprise reactive co-monomer that may or may not
be water-soluble. The molar fraction of the reactive co-monomer may
range, for example, from about 0.001 to 10, from about 0.01 to 10,
from about 0.001 to 1, or from about 0.01 to 1. In some instances,
a copolymer of
poly(N,N-dimethylacrylamide-co-pentafluorophenylacrylate) may be
used to form the hydrogel. In other instances, copolymers that
contain bromoacetamidepentylacrylamide (BRAPA) may be used either
alone or in conjunction with other polymers to form the
hydrogel.
An example of a hydrogel coupled to a silicon dioxide support is
shown in FIG. 19. The hydrogel may provide for a physically
entangled polymer network. The physical entanglement may restrict
the thermodynamic extension and collapse to a certain degree. The
presence of the charged nucleic acid molecules and their counter
cations and other ions distributed throughout the hydrogel may
prevent resistivity domains from forming. The use of hydrogels may
improve the effective loading density of biomolecules and may be
flexible enough to allow other molecules, such as enzymes, to
penetrate to perform enzymatic reactions. In some examples, the
surface density of a fifteen base primer may range from
2.0.times.10.sup.5 to 1.2.times.10.sup.6, which may be an order of
magnitude higher than what may be observed with magnetic beads
(see, e.g., FIG. 18).
The hydrogel may be seeded with primer oligos. The hydrogel may be
seeded with between about 1 picomolar (pM) and 10,000 pM, between
about 1 pM and about 8,000 pM, between about 1 pM and about 6,000
pM, between about 1 pM and 4,000 pM, between about 1 pM and 2,000
pM, between about 1 pM and 1,000 pM, between about 1 pM and 500 pM,
or between about 1 pM and 250 pM primer oligos. In some
embodiments, the hydrogel is seeded with greater than 1 pM, greater
than 10 pM, greater than 50 pM, greater than 100 pM, greater than
500 pM, greater than 1,000 pM, greater than 2,000 pM, greater than
4,000 pM, greater than 6,000 pM, greater than 8,000 pM, greater
than 10,000 pM, or more primer oligos. In some embodiments, the
hydrogel is seeded with less than about 10,000 pM, less than about
8,000 pM, less than about 6,000 pM, less than about 4,000 pM, less
than about 2,000 pM, less than about 1,000 pM, less than about 500
pM, less than about 100 pM, less than about 50 pM, less than about
10 pM, less than about 5 pM, less than about 1 pM, or fewer primer
oligos. The primers may be reactive with the hydrogel. The primer
may contain a reactive nucleophile. The reactive nucleophile may be
a thiol. For example, a primer comprising a benzylthiol group may
be an effective nucleophile.
FIG. 20 shows a structural drawing of a hydrogel comprised at least
partially of BRAPA. The BRAPA may serve as a branching agent during
atom-transfer radical-polymerization and as reactive group for
biomolecule conjugation. The BRAPA may copolymerizes with
acrylamide and other vinyl containing monomers to form a thin film
hydrogel on the support surface. The residual bromoacetamide groups
react with a variety of nucleophiles, including thiolated
oligonucleotides. This reaction is exemplified by the benzthiolated
Primer B conjugating with the bromoacetamide pendent group of the
polymer chain.
The polymeric material may be a porous polymer monolith (PPM). The
PPM may be a homopolymer, copolymer, or terepolymer that comprises
reactive functional groups. The reactive functional groups may be
reactive to a variety of chemical moieties including, but not
limited to, primary amines, secondary amines, mercaptos, hydroxyl
groups, thiol groups, and carboxylic acid groups. The reactive
functional groups may be comprised of carboxylic acids, esters of
N-hydroxysuccinimide, pendants of 4,4'-dimethylazlactone, or esters
of tetrafluorophenol and pentafluorophenol.
The PPM may be polymerized using a variety of approaches, including
chemical, thermal, or UV radiation approaches. FIGS. 21A-21D and
FIG. 22 show the procedure for fabricating a support with a PPM
thin film. FIG. 21A shows the substrate surface, previously cleaned
by isopropanol rinsing and soaking in RCA solutions. FIG. 21B shows
the substrate surface modified by (3-acryloxypropyl)
trimethoxysilane, which may introduce polymerizable function groups
to the surface. FIG. 21C shows a solution of reactive monomers,
pentafluorophenyl acrylate or vinylazlactone for example, a
multifunctional crosslinker, and initiators in a porogen solvent
dispensed on the surface of the substrate in preparation for
polymerization. Photoinitiators may be used for initiating the
polymerization reaction. The photoinitiators may be biomolecular
photoinitiators that may comprise benzophenone and
.alpha.,.alpha.-dimethoxy-.alpha.-phenylacetophenone. This mixture
of initiators may enable photopolymerization to proceed in open
air. In some cases, a glass lid having a fluorinated surface as a
release may be placed over the monomer mixture prior to
polymerization. FIG. 21D shows the support with PPM thin film.
After polymerization the lid is released and removed and the PPM
film is rinsed with hexane and air dried to give a reactive thin
film of PPM for bioconjugation.
An illustrative example for the preparation of PPM grafted onto the
substrate surface is shown in FIG. 22. Briefly, a silicon dioxide
substrate with a polymerizable surface moiety may be exposed to a
crosslinker, porogen, and initiators and treated with UV exposure
followed by washing to form a PPM film covalently attached to the
substrate surface. The PPM film may then be reactive and used for
bioconjugation.
The PPM may be seeded with primer oligos. The PPM may be seeded
with between about 1 picomolar (pM) and 10,000 pM, between about 1
pM and about 8,000 pM, between about 1 pM and about 6,000 pM,
between about 1 pM and 4,000 pM, between about 1 pM and 2,000 pM,
between about 1 pM and 1,000 pM, between about 1 pM and 500 pM, or
between about 1 pM and 250 pM primer oligos. In some embodiments,
the PPM is seeded with greater than 1 pM, greater than 10 pM,
greater than 50 pM, greater than 100 pM, greater than 500 pM,
greater than 1,000 pM, greater than 2,000 pM, greater than 4,000
pM, greater than 6,000 pM, greater than 8,000 pM, greater than
10,000 pM, or more primer oligos. In some embodiments, the PPM is
seeded with less than about 10,000 pM, less than about 8,000 pM,
less than about 6,000 pM, less than about 4,000 pM, less than about
2,000 pM, less than about 1,000 pM, less than about 500 pM, less
than about 100 pM, less than about 50 pM, less than about 10 pM,
less than about 5 pM, less than about 1 pM, or fewer primer oligos.
The primers may contain a variety of reactive groups that are
reactive with the PPM. Example reactive groups include primary and
secondary amines, mercaptos, hydroxyl groups, thiol groups, and
carboxylic acids.
Primer density may be determined by a hybridization-dehybridization
(Hyb-DeHyb) assay and PicoGreen visualization. FIG. 23 shows a
schematic of the Hyb-DeHyb assay performed with a surface
conjugated primer and a FAM-labeled oligo that is complementary to
the primer. FIG. 24 illustrates the process flow for preparing a
primer conjugated support and the techniques for determining primer
conjugation.
Polymer grafting may be achieved both with and without the use of a
silane to activate the substrate surface. FIG. 25 shows improved
primer density of multiple substrate surfaces prepared both with
and without the use of a silane. Primer density may be found to be
higher in the absence of a silane. In the absence of the silane
group, rather than the polymeric material forming an amide bond
with the aminosilane coupling agent, the polymeric material may
couple directly to the support surface through ester bond
formation. It is unexpected to achieve a higher primer density
result from the absence of the aminosilane coupling agent and that
the ester linkages survive multiple 100 millimolar (mM) NaOH
washes.
The primer may participate in an amplification reaction. During the
amplification reaction, the primer may form clonal colonies or the
primer may cover the entire surface of the support and form a
lawn-type coverage. The primer may be seeded at a concentration
ranging from 1 pM to 4000 pM. The seeding density may be critical
for the formation of clonal colonies. For example, if the seeding
density is too high and primers are too close together a lawn-type
coverage will form of mixed colonies, which may not permit the use
of a confined amplification approach. If the seeding density is too
low, the support will not be utilized effectively. After seeding,
the templates may undergo an amplification reaction. A variety of
amplification reactions may be used, but methods of choice may
include methods that use a confined amplification scheme or
amplification schemes that may be combined with a confinement
approach. Examples of amplification schemes include, polymerase
chain reaction, recombinase polymerase amplification, invader
amplification, bridge amplification, and wildfire
amplification.
The immobilized primer arrangement with regard to the polymeric
coated support and an example use of confinement in the system is
shown in FIGS. 26A-26C. FIGS. 26A and 26B show a schematic for
invader amplification, a surface confined amplification method.
Briefly, a non-extendable invader may facilitate opening of the
duplex of a target nucleic acid in close proximity to the support
surface. Opening the duplex of the target nucleic acid may allow
for hybridization of the template with a surface immobilized
primer, which may lead to a primer extension and amplification.
FIG. 26C shows the template density after amplification, as a
function of starting template concentration, from a concentration
titration experiment. In this example, a seeding concentration from
3 pM to 4000 pM is used. From 40 pM to 4000 pM the system may be
saturated (i.e., a lawn of templates is expected to be formed) and
little change in amplification level is observed. At lower seeding
concentrations the amplification level may drop off, which may be
explained by the formation of distinct colonies.
Primer seeding density and amplification may be visualized using
FAM-PicoGreen visualization. FIG. 27 shows an example of
FAM-PicoGreen visualization both after primer seeding and after
amplification.
The sequencing reaction may generate an electrochemical signal. The
signal may be indicative of individual nucleic acid bases. The
electrochemical signal may include impedance, potentiometric,
cyclic voltammetric, spectroscopic, or amperometric signal. In
other cases, the signal may be monitored using non-electrochemical
techniques. Non-electrochemical techniques may include fluorescent
spectroscopy, surface plasmon resonance, or cantilever
techniques.
The electrodes may be coupled to a Debye layer (or double layer)
during sequencing. The nucleic acid to be sequenced may also be
within the Debye layer so that the sequencing reaction may occur
within the layer. Incorporation of nucleotides into the nucleic
acid molecules within the Debye layer may generate a measureable
signal. The measureable signal may be an electrochemical signal or
an impedance signal. The signal may be measured during a transient
or steady state condition.
Primer extension may be monitored via electronic detection. FIG. 28
shows an example result of electronic detection of primer extension
and nucleotide incorporation for nucleic acid templates amplified
on a support surface. The nucleic acid templates amplified on the
support surface may be bound to a primer and polymerase and exposed
to a solution containing all four nucleotides. The solution
containing all four nucleotides is called the Run-Off mix. FIG. 28
shows the jump in the electronic signal following introduction of
the Run-Off mix onto the support. The multiple traces in FIG. 28
may denote the jumps in electronic signal observed for different
sets of sensors or jump signals following correction with a
reference or background signal. The jump in electronic signal may
demonstrate primer extension due to nucleotide incorporation.
Primer extension may also be verified via capillary
electrophoresis. FIG. 29 shows the capillary electrophoresis
readout of the extension of a FAM-labeled primer during a run-off
or sequencing experiment. The extended strand may be melted off
using sodium hydroxide following the run-off or sequencing reaction
and may be run on a capillary electrophoresis. Four supports (191A,
191B, 191C, and 191D) are extended using run-off or sequencing and
show complete extension. The size of the extended strand is larger
than the size of the 120 base pair control, which is expected based
on the size of the amplification template. The smaller peaks
present may be close to the noise level of the system.
An example of monitoring the support from conjugation of the primer
to run-off or sequencing experiments is shown in FIGS. 30A and 30B.
FIG. 30A shows FAM-PicoGreen micrographs both after primer
conjugation and after target amplification. FIG. 30B shows the
cumulative FAM-PicoGreen micrograph and the electronic signal due
to the run-off or sequencing experiment.
Methods for Redox Mediated Sequencing
A redox mediator moiety can be any species that comprises one or
more components (e.g., functional groups) that can participate in a
redox reaction, such as, for example, reduction or oxidation. In an
oxidation reaction, a redox mediator moiety donates one or more
electrons to another species, such that the redox mediator moiety
loses the one or more elections to another species, resulting in an
oxidized redox mediator moiety. Moreover, in a reduction reaction,
a redox mediator moiety can accept one or more electrons from
another species, such that the redox mediator moiety gains the one
or more electrons from the other species, resulting in a reduced
redox mediator moiety. In some cases, a redox mediator moiety can
donate an electron(s) in an oxidation reaction, but cannot readily
gain an electron(s) from another species in a reduction reaction.
In other cases, a redox mediator moiety can receive an electron(s)
in a reduction reaction, but cannot readily donate an electron(s)
to another species in an oxidation reaction. Still, in other cases,
a redox mediator moiety can both donate an electron(s) to another
species in an oxidation reaction and gain an electron(s) from
another species in a reduction reaction. Moreover, the charge of a
redox mediator moiety will vary depending upon its oxidation state.
Depending upon its oxidation state, a redox mediator moiety may be
positively charged, neutral or negatively charged.
The cycle of oxidation and/or reduction of a given redox mediator
moiety and its oxidized and/or reduced forms can occur cyclically
such that an electron(s) is repeatedly transferred back-and-forth
between a redox mediator moiety and another species. For example,
in the case of a redox mediator moiety that donates an electron(s)
in an oxidation reaction, the redox mediator moiety can donate its
electron(s) to an additional species to form an oxidized redox
mediator moiety and a reduced additional species. The oxidized
redox mediator moiety can then receive an electron(s) from the
reduced additional species in a reduction reaction, thus,
regenerating the redox mediator moiety. In another example, in the
case of a redox mediator moiety that accepts an electron(s) from an
additional species in a reduction reaction, the redox mediate
moiety can accept an electron(s) from the additional species to
form a reduced redox mediator moiety and an oxidized additional
species. The reduced redox mediator moiety can then donate an
electron(s) back to the oxidized additional species to regenerate
the redox mediator moiety. In the case of a redox mediator moiety
that can readily donate and accept an electron(s), both types of
cycling can occur. During cycling of an electron(s) between
species, a redox mediator moiety may go from neutral to negatively
charged or vice versa; from positively charged to neutral or vice
versa; from one positive charge to a higher positive charge (e.g.,
+1 to +2); from one negative charge to a higher negative charge
(e.g., -1 to -2); from one positive charge to a lower positive
charge (e.g., +2 to +1); or from one negative charge to a lower
negative charge (e.g., -2 to -1).
A redox mediator moiety can comprise an organic compound, an
organometallic compound, a nanoparticle, one or more metals,
another suitable material and combinations thereof. In some cas
References